Silk-derived protein for treating inflammation

ABSTRACT

Described herein are methods for reducing inflammation by administration of an effective amount of silk-derived proteins (SDP) or a fraction thereof to a subject having an inflammatory condition. The methods include the treatment of inflammatory conditions and wounds, including corneal wounds, comprising the topical administration of an effective amount of SDP material as described herein.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/324,844 filed on Feb. 11, 2019, now granted U.S. Pat. No. 11,242,367,which is a national stage entry under 35 U.S.C. § 371 of InternationalPatent Application No. PCT/US2017/046659 filed Aug. 2, 2017, whichclaims priority under 35 U.S.C. § 119(e) to U.S. Provisional PatentApplication Nos. 62/374,532 filed Aug. 12, 2016, 62/407,863 filed Oct.13, 2016, and 62/467,697 filed Mar. 6, 2017, which applications areincorporated herein by reference.

GOVERNMENT SUPPORT

This invention was made with government support under Grant No. 1152561awarded by National Science Foundation and Grant No. W81XWH-15-C-0150awarded by the United States Army. The government has certain rights inthe invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Oct. 2, 2017, isnamed 114_014WO1_SL Sequence Listing and is 3,246 bytes in size.

BACKGROUND OF THE INVENTION

Inflammation describes the cooperative response of afflicted cells toharmful stimuli (e.g., infection) or local tissue injury in attempt torestore homeostasis. Exposure of resident cells to these aberrantconditions initiates intracellular signaling cascades that result in theproduction and secretion of inflammatory mediators. Localized depositionof these inflammatory entities serves to recruit immune cells (e.g.,neutrophils) from the interstitium and vasculature to the site of injuryor insult. Successful removal of the stimulus is followed by tissuerepair, which introduces new immune cell types (e.g., macrophages) andsignaling intermediaries and concludes the acute inflammatory response(Medzhitov, Nature, 2008. 454(7203): 428-435). However, if tissuehomeostasis is not achieved within this timespan, a chronic inflammatoryresponse ensues, whereby additional immune cells are introduced to thesite of injury in attempt to contain it. Nevertheless, chronicinflammation can permanently undermine the healthy tissue state.Dysregulated signaling pathways that result from chronic inflammationhave been implicated in a multitude of diseases, including dry eye,autoimmune disorders, cardiovascular disease, and cancer.

While the instigating cause of the immune response can be foreign to thehost, disruption of localized tissue homeostasis due to aberrant cellsignaling can also generate concentration gradients of signalingmolecules that drive immune cell recruitment and response. For example,disruptions in tear film composition at the apical surface of the eyeresults in the increased production of proinflammatory cytokines thatstimulate the immune cascade both acutely and chronically (Luo et al.,Eye & Contact Lens, 2005. 31(5): 186-193). This condition, known askeratoconjunctivitis sicca or dry eye syndrome (DES), persists due to aconstant inflammatory stimulus that translates into altered cellularmechanical stress (via cell shrinkage) and gene expression (Brocker etal., Biomolecular Concepts, 2012. 3(4): 345-364). This further lends tothe production of cytokines, which act on the local microenvironment andrecruit mediator cell types of the acute inflammatory response. In turn,migratory neutrophils secrete additional pro-inflammatory morphogensthat alter ocular limbal vascular permeability and thereby permit influxof activated T cells to the irritated eye surface, transitioning to achronic inflammatory state (Baudouin, Survey of Ophthalmology, 2001.45(2): S211-220).

One specific example of such a stimulus occurs with tear film fluidhyperosmolarity, which is caused by accelerated tear evaporation or teargland hyposecretion. If the hyperosmotic stimulus is not addressed bythe actions of immune cell mediators, homeostasis is not achieved anddestruction of the ocular surface and tear glands evolves over timethrough dysregulated tissue remodeling mechanisms of the ocular surface.This cascade can lead to the increased production of matrixmetalloproteinase 9 (MMP-9) that degrades the ocular surface in arunaway feed-forward mechanism of tissue remodeling.

Approaches to mitigate the inflammatory response typically target theproduction of pro-inflammatory signaling molecules. These include theuse of glucocorticoid steroids, which function to decrease production ofproinflammatory proteins while simultaneously increasing production ofanti-inflammatory proteins within a recipient cell (Rhen et al., The NewEngland Journal of Medicine, 2005. 353(16): 1711-1723). However, theeffects of glucocorticoid signaling are potent and not confined toimmune cell signaling, with impacts on metabolic and fluid homeostasis,neuronal function, and fetal development. Therefore, glucocorticoidsignaling is heavily regulated and generally restricted to chronichyperactive immune system disorders. Conversely, non-steroidalanti-inflammatory drugs (NSAIDs), which include aspirin, ibuprofen, andnaproxen, function to inhibit cyclooxygenase (COX) enzyme activity,which precedes prostaglandin production that is heavily increased ininflamed cells (Ricciotti et al., Arteriosclerosis, Thrombosis, andVascular Biology, 2011. 31(5): 986-1000). NSAIDs are effectivecombatants of the inflammatory process, but are typically administeredsystemically and inhibit the functions of COX enzymes elsewhere in thebody, which can contribute to stomach ulcerations and renal dysfunction.Given the off-target side effects of the above-mentioned therapeuticstrategies, the anti-inflammatory agent ideally should be localized tothe injured or infected tissue (e.g., skin, or eye surface).

The application of targeted anti-inflammatory therapies offers promiseto attenuate the immune cell response with minimal side effects. Forexample, the development of antagonist antibodies againstpro-inflammatory mediators (e.g., chemokines) has been employed forinflammatory diseases with promising efficacy (Skov et al., Journal ofImmunology, 2008. 181(1): 669-679). However, the production cost ofthese proteins is significant and variability in antibody production mayinfluence therapeutic efficacy. Alternatively, pharmacologicalinhibitors of signaling pathways upstream of chemokine production and/orsecretion would be theoretically ideal, since they would eliminaterecruitment of immune cell types involved in the acute and eventualchronic inflammatory response. Among these theoretical targets would bethe nuclear factor-kappa B (NF-κB) transcription factor family, which isheavily implicated in the production of acute pro-inflammatorymorphogens (Hayden et al., Cell Research, 2011. 21(2): 223-244).Natively, NF-κB subunits reside in the cytoplasm and are prevented fromnuclear translocation by the masking of protein residues that targetdelivery to this region. However, upon stimulation, the inhibitoryprotein is quickly degraded, thereby allowing translocation and DNAbinding of NF-κB proteins and subsequent gene transcription.

A number of natural and synthetic inhibitors of NF-κB exist. Among theformer is silk fibroin, which is a dimer composed of heavy and lightprotein chains (390 kD and 26 kD, respectively) isolated from thesilkworm cocoon (reviewed by Altman et al., Biomaterials, 2003. 24(3):401-416). These globular proteins assemble into a fibrillar architectureby the disulfide linkage of light and heavy chains and exhibitremarkable homogeneity in β-sheet secondary structure. Fibroin has beenshown to inhibit transcription and upstream activation (i.e., viainhibition of protein kinases) of NF-κB protein subunits (Chon et al.,International Journal of Molecular Medicine, 2012. 30(5): 1203-1210).Furthermore, hydrolyzed peptide fragments of fibroin have been shown toinhibit transcription of proinflammatory molecules that are classicallyunder control of NF-κB (Kim et al., J. Neurosurg., 2011. 114(2): 485-90;J. Microbiol. Biotechnol., 2012. 22(4): 494-500). However, the use ofsilk fibroin has not resulted in effective treatments for inflammatoryconditions and wounds.

Furthermore, eye disease and injury remain persistent and seriousconcerns to the general world population. Ocular disease and trauma posean immediate threat to normal vision by extending throughout the healingprocess and risking permanent disability or blindness from prolongedinfection, chronic inflammation, and scar formation. As such, there isan immediate need for therapies to reduce inflammation and acceleratehealing of the injured or inflamed ocular tissue.

SUMMARY

The invention provides a modified silk fibroin protein for therapeuticapplications such as reducing inflammation as well as promoting woundhealing and tissue regeneration. The modified protein has been shown tosupport corneal epithelial cell attachment and proliferation. Thesilk-derived protein (SDP) described herein is a fibroin-derived proteincomposition that has reduced beta-sheet activity, resulting in ahighly-soluble and aqueous-stable material. SDP can be readilyincorporated into solution-based product formulations at highconcentrations. Another advantage is that SDP has a high level ofmiscibility with other dissolved ingredients, such as those typicallyincluded in an ophthalmic formulation. One specific use of SDP is itsinclusion in ophthalmic formulations as a novel protein component toenhance solution-wetting characteristics on the ocular surface. The SDPcan be fractionated and it was surprisingly discovered that lowmolecular weight fractions of SDP have enhanced anti-inflammatoryproperties.

The invention therefore provides a fibroin-derived protein compositionthat possesses enhanced stability in an aqueous solution, wherein theprimary amino acid sequences of the fibroin-derived protein compositiondiffer from native fibroin by at least 4% with respect to the absolutevalues of the combined differences in amino acid content of serine,glycine, and alanine; cysteine disulfide bonds between the fibroin heavyand fibroin light protein chains of fibroin are reduced or eliminated; aplurality of peptide chains in the protein composition terminate inamide (—C(═O)NH₂) groups; the composition has a serine content that isreduced by greater than 25% compared to native fibroin protein, whereinthe serine content is at least about 5%; and wherein the averagemolecular weight of the fibroin-derived protein composition is less than40 kDa and greater than 2 kDa.

In some embodiments, greater than 50% of the protein chains of theprotein composition have a molecular weight within the range of 10 kDato 60 kDa. In various embodiments, the protein composition does not gelupon sonication of an aqueous solution of the protein composition atconcentrations of up to 10% w/w.

The protein composition can have less than 8% serine, less than 7%serine, or less than 6% serine amino acid residues. The proteincomposition can have greater than 46% glycine amino acids, greater than46.5% glycine amino acids. The protein can have greater than 30% alanineamino acids, or greater than 30.5% alanine amino acids.

The protein composition can completely re-dissolves in water after beingdried to a thin film. Beta-sheet protein structures are minimal orabsent in aqueous solution. The protein composition can maintain anoptical absorbance in aqueous solution of less than 0.25 at 550 nm afterat least five seconds of sonication.

The invention also provides an ophthalmic formulation comprising theprotein composition described herein, and water, and optionally one ormore of a buffering medium, a salt, a stabilizer, a preservative, and alubricant.

The invention further provides a method for reducing inflammationcomprising administering a fibroin-derived protein composition toinflamed tissue; wherein the primary amino acid sequences of thefibroin-derived protein composition differ from native fibroin by atleast 4% with respect to the absolute value of the combined differencesin amino acid content of serine, glycine, and alanine; cysteinedisulfide bonds between the fibroin heavy and fibroin light proteinchains of fibroin are reduced or eliminated; a plurality of peptidechains in the protein composition terminate in amide (—C(═O)NH₂) groups;the composition has a serine content that is reduced by greater than 25%compared to native fibroin protein, and wherein the serine content is atleast about 5%; and wherein the average molecular weight of thefibroin-derived protein composition is less than 60 kDa and greater than2 kDa; thereby reducing transcription factor signaling within cellnuclei of the tissue, thereby reducing the inflammation. The averagemolecular weight of the fibroin-derived protein composition can also beless than 55 kDa, and/or greater than about 5 kDa, greater than 10 kDa,greater than 15 kDa, or greater than 20 kDa.

The administration to inflamed tissue can reduce transcription of one ormore of the inflammatory genes TNF-α, MMP-9, IL-1β, and IL-6. Thereduction can be as much as 20%, 40%, 50%, or 60% compared to in absenceof the protein composition. The administration can be to the cornea andthe administration can reduce the presence of MMP-9 in the cornea. Theadministration can be to the eye and the administration reducesinflammation on the ocular surface, for example, as determined by ELISAmeasurement of proinflammatory markers in the tear film. The thereduction in inflammation can be accompanied by an increase in cellmigration rates at the point of inflammation, for example, an increasein cell proliferation, as determined by an MTT assay.

The protein composition can have an average molecular weight less than40 kDa, or less than 35 kDa. The fibroin-derived protein composition canbe dissolved in an ophthalmic formulation comprising one or more of abuffering medium, a salt, a stabilizer, a preservative, and a lubricant.

The inflammation can be inflammation caused by an ocular condition,wherein the ocular condition is dry eye syndrome, corneal ulcer, cornealerosion, corneal abrasion, corneal degeneration, corneal perforation,corneal scarring, epithelial defect, keratoconjunctivitis, idiopathicuveitis, corneal transplantation, age-related macular degeneration,diabetic eye, blepharitis, glaucoma, ocular hypertension, post-operativeeye pain and inflammation, posterior segment neovascularization,proliferative vitreoretinopathy, cytomegalovirus retinitis,endophthalmitis, choroidal neovascular membrane, vascular occlusivedisease, allergic eye disease, tumor, retinitis pigmentosa, eyeinfection, scleritis, ptosis, miosis, eye pain, mydriasis, neuralgia,cicatrizing ocular surface disease, ocular infection, inflammatoryocular disease, ocular surface disease, corneal disease, retinaldisease, ocular manifestations of systemic diseases, hereditary eyecondition, ocular tumor, increased intraocular pressure, herpeticinfection, ptyrigium or scleral tumor, wound sustained to ocularsurface, post-photorefractive keratotomy eye pain and inflammation,thermal or chemical burn to the cornea, scleral wound, or keratoconusand conjunctival wound. In one embodiment, the inflammation is caused bydry eye syndrome.

The invention further provides for the use of a fibroin-derived proteincomposition described herein for treating inflammation, wherein theprimary amino acid sequences of the fibroin-derived protein compositiondiffer from native fibroin by at least 4% with respect to the absolutevalue of the combined differences in amino acid content of serine,glycine, and alanine; cysteine disulfide bonds between the fibroin heavyand fibroin light protein chains of fibroin are reduced or eliminated; aplurality of peptide chains in the protein composition terminate inamide (—C(═O)NH₂) groups; the composition has a serine content that isreduced by greater than 25% compared to native fibroin protein, andwherein the serine content is at least about 5%; and wherein the averagemolecular weight of the fibroin-derived protein composition is less than60 kDa and greater than 10 kDa. The protein composition can have anaverage molecular weight less than 35 kDa. The composition can be acomposition for the treatment of dry eye syndrome.

Accordingly, SDP compositions are provided herein that possess enhancedstability in aqueous solutions in which the primary amino acid sequenceof native fibroin is modified from native silk fibroin, wherein cysteinedisulfide bonds between the fibroin heavy and fibroin light proteinchains reduced or eliminated; wherein the composition has a serinecontent that is reduced by greater than 40% compared to native fibroinprotein; and wherein the average molecular weight of the SDP is lessthan about 60 kDa.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the specification and are includedto further demonstrate certain embodiments or various aspects of theinvention. In some instances, embodiments of the invention can be bestunderstood by referring to the accompanying drawings in combination withthe detailed description presented herein. The description andaccompanying drawings may highlight a certain specific example, or acertain aspect of the invention. However, one skilled in the art willunderstand that portions of the example or aspect may be used incombination with other examples or aspects of the invention.

FIG. 1A-D. p65 protein immunostaining (white) of hCLE cultures for NF-κBactivation. (A) Negative control cultures treated with PBS showedcytosolic p65 staining indicating native NF-κB inactivity. (B) Positivecontrol cultures treated with PBS containing 1 ng/mL TNF-α demonstratedpunctate p65 nuclear staining indicating protein translocation and hencea high level of NF-κB activation. (C and D) Culture treated with PBS,TNF-α, and 0.1% SDP or 1% SDP demonstrated a dose-dependent reduction innuclear p65 staining indicating higher SDP concentrations inhibit NF-κBactivation to a greater extent, respectively. (Scale bars=20 μm).

FIG. 2. Summary qPCR results of relative fold gene expression for TNF-αand MMP-9 for hCLE cultures treated with PBS, PBS plus 0.5% SDP, PBSplus 1 ng/mL TNF-α cytokine, and PBS plus 1 ng/mL TNF-α cytokine plus0.5% SDP. TNF-α and MMP-9 are known genetic markers of NF-κB activation.Cultures stimulated with TNF-α and treated with 0.5% SDP were found tohave a 6-fold reduction in gene expression relative to TNF-α cytokinestimulated controls (Δp<0.01 compared to PBS for respective GOI; Θp<0.01vs. SDP for respective GOI; and #p<0.05 vs. indicated groups; n=3).

FIG. 3A-E. (A) Representative cross-section image of corneal tissueobtained from native rabbits immunostained for MMP-9. (B-D)Representative immunohistochemical images of corneal cross-sectionsobtained from rabbits harvested 72-hours post-surgery for the varioustreatment groups. MMP-9 staining decreased for both SDP treated groups(C and D) when compared to PBS-treated animals (B). (Scale bar=50 μm).(E) Summary graph of measured staining intensity (fluorescenceintensity) of MMP-9 in corneas treated with PBS, 0.5% SDP, or 2% SDP(*p<0.01 vs. Control; ^(#)p<0.01 compared to 0.5% SDP, n=3).

FIG. 4. qPCR results of relative fold gene expression of IL-1β and IL-6for rabbit corneas treated with PBS, PBS plus 0.5% SDP, and PBS plus 2%SDP over a 72-hour period following surgical denudement of theepithelial surface. IL-1β and IL-6 are known genetic markers ofinflammation within the corneal tissue environment. Expression of bothmarkers was significantly reduced in the presence of SDP treatment(*p<0.01 vs. PBS for each GOI; n=6).

FIG. 5. Summary graph of H₂O₂ levels measured by electron paramagneticresonance (EPR) spectroscopy in the presence of defined concentrationsof dissolved proteins (PASF, SDP or SDP-4). H₂O₂ (20 μM) was incubatedin the absence (Control) or presence of PASF, SDP, or SDP-4 (each at0.5%, 1%, or 5%), and then introduced to a H₂O₂-specific spin probe. EPRsignal generated by the oxidized spin probe for each sample was measuredand normalized to control samples (i.e., lacking protein). PASFincreased EPR signal amplitude with increasing protein concentration. Incontrast, SDP evoked a concentration-dependent reduction in EPR signalamplitude, demonstrating that SDP proteins scavenge H₂O₂. H₂O₂scavenging was even more robust in the presence of SDP-4 proteins. Errorbars are represented as S.D., N=3.

FIG. 6. SDS-PAGE lanes 2-5 represent the respective molecular weight(MW) distributions of SEC-fractionated SDP populations for whichbiological impact was evaluated (SDP-1, SDP-2, SDP-3, SDP-4, and SDP).Lane 6 illustrates the non-fractionated SDP distribution from whichfractions were derived. MW standards are shown in lane 1.

FIG. 7. Representative images from in vitro wound healing assaysdemonstrate that cell growth and migration into the cell-free region(wound), outlined in white, is significantly accelerated in the presenceof 5-mg/mL SDP-3 or SDP-4.

FIG. 8. Summary bar graph illustrating percent wound closure atindicated time points during the scratch wound assay (*p<0.05 vsControl), (#p<0.05 vs SDP-1, n=3), (†p<0.05 vs SDP-2, n=3).

FIG. 9. MTT analysis of epithelial cell viability in hCLE culturestreated with 5-mg/mL of fractionated SDPs or (saline buffer) control.Treatment with SDP-3 and SDP-4 significant increased cell proliferationrelative to control cells. Treatment with SDP-1 or SDP-2 did not changecell proliferation relative to controls (*p<0.05 vs. Control, n=3;#p<0.05 vs. SDP-1, n=3; †p<0.05 vs. SDP-2, n=3).

FIG. 10. qPCR summary of TNF-α, MMP-9, and Interleukins-1α/β, -6, and -8transcription in hCLE cells untreated (native) or stimulated with TNF-αto initiate inflammatory signaling, and treated with 1 mg/mL offractionated SDP. Treatment with SDP-3 and SDP-4 significantly decreasedtranscription of the defined inflammatory genes, relative to controlcells treated with PBS. (†p<0.05 vs Native, n=3; *p<0.05 vs Control,n=3).

FIG. 11. ELISA analysis of TNF-α cytokine secretion by hCLE cellsuntreated (native) or stimulated with TNF-α to initiate inflammatorysignaling, and treated with 1 mg/mL of SDP fractions. Treatment withSDP-3 and SDP-4 significantly decreased secretion of thepro-inflammatory cytokine TNF-α, while SDP-2 significantly increasedsecretion, relative to control cells treated with PBS. (†p<0.05 vsNative, n=3), (*p<0.05 vs Control, n=3).

FIG. 12. Summary of Transwell migration assay demonstrating thattreatment with TNF-α significantly increased HL-60 inflammatory cellmigration relative to untreated (native) cultures. Addition of SDP-4 (1mg/mL) resulted in a significant reduction of TNF-α driven HL-60 cellmigration (†p<0.05 vs Control, n=3; *p<0.05 vs Native, n=3).

DETAILED DESCRIPTION OF THE INVENTION

The invention provides protein compositions derived from SDP fortreating inflammation and for treating wounds. Evidence supports thatproteins isolated from the silkworm cocoon stimulate growth of cornealcells and alter expression of genes implicated in wound healing andinflammation (FIGS. 1-5). The protein compositions described herein alsopossess enhanced solubility and stability in aqueous solutions. Methodsof making protein compositions include modifying the primary amino acidsequence of native fibroin such that cysteine disulfide bonds betweenthe fibroin heavy and fibroin light protein chains are reduced oreliminated. Additionally, the serine content of the protein compositionis reduced by greater than 40% compared to native fibroin protein, andthe average weight molecular weight of the proteins is less than about60 kDa. In some cases, protein compositions described herein include orbe derived from the protein compositions described in U.S. Pat. No.9,394,355, the entire disclosure of which is hereby incorporated byreference into this specification. Lower average molecular weightfractions can also be isolated to provide compositions with enhancedanti-inflammatory activity by virtue of their enhanced ability to reducethe expression of pro-inflammatory genes compared to larger molecularweight fractions or the SDP composition in its entirety.

Discrete SDP subpopulations further enhance healing and reduceinflammation in the body, particularly in corneal tissue. Selected SDPfractions have been shown to enhance the effective potency of SDP oncell migration response and inflammation. The SDP fractions wereprepared by extracting Bombyx mori silkworm cocoons fibers in 0.3%sodium carbonate at 95° C., and then fibroin fiber was dissolved in 54%LiBr solution. The dissolved solution was autoclaved, coarse filtered,and then purified by diafiltration. The material was then filteredthrough a nominal polypropylene filter to produce a final SDP solution.The SDP solution was then separated by molecular weight (MW) through theuse of one of two methods depending on the specific experiment. In thefirst method, centrifugation using molecular weight cutoff filters wasutilized to separate out SDP protein fractions by molecular weightcutoff (MWCO) size. For example, SDP can be centrifuged at 5000×g untilsamples are reduced to 10% of starting volume (e.g., 15 mL initialvolume concentrated to 1.5 mL, for certain experiments describedherein). Proteins sieved through the filter are less than the molecularMWCO of a particular filter; the retained proteins are generally ofequal or greater molecular weight. In a second method, sample SDPfractions can also be isolated by size exclusion chromatography (SEC) toproduce discrete protein sub-populations, or fractions. Four fractionsof decreasing average molecular weight were produced and are referred toas SDP-1, SDP-2, SDP-3, and SDP-4 (FIG. 6).

The two smallest molecular weight SDP fractions, SDP-3 and SDP-4,significantly reduce inflammation and enhance wound healing of hCLEcultures in vitro through increased cell migration and proliferationeffects (FIG. 7-12). These SDP fractions inhibit inflammatory signaling,which can further enhance wound healing and improve long-term patientoutcomes. The protein fractions derived from SDP can therefore be usedfor treating inflammation and related conditions. One specifictherapeutic application is in the treatment of dry eye disease, which isknown to be an inflammatory related disease that is driven, in part, bythe NF-κB signaling pathway, which is inhibited by SDP. In anotherspecific therapeutic application, SDP may be utilized to treatpost-surgical injuries to induce enhanced healing outcomes by reducinginflammation and/or increasing cell proliferation and/or migration, suchas those injuries produced during refractive eye surgery or cataractremoval, and/or accidental injuries where the corneal epithelium iscompromised.

Definitions

The following definitions are included to provide a clear and consistentunderstanding of the specification and claims. As used herein, therecited terms have the following meanings. All other terms and phrasesused in this specification have their ordinary meanings as one of skillin the art would understand. Such ordinary meanings may be obtained byreference to technical dictionaries, such as Hawley's Condensed ChemicalDictionary 14^(th) Edition, by R. J. Lewis, John Wiley & Sons, New York,N.Y., 2001.

References in the specification to “one embodiment”, “an embodiment”,etc., indicate that the embodiment described may include a particularaspect, feature, structure, moiety, or characteristic, but not everyembodiment necessarily includes that aspect, feature, structure, moiety,or characteristic. Moreover, such phrases may, but do not necessarily,refer to the same embodiment referred to in other portions of thespecification. Further, when a particular aspect, feature, structure,moiety, or characteristic is described in connection with an embodiment,it is within the knowledge of one skilled in the art to affect orconnect such aspect, feature, structure, moiety, or characteristic withother embodiments, whether or not explicitly described.

The singular forms “a,” “an,” and “the” include plural reference unlessthe context clearly dictates otherwise. Thus, for example, a referenceto “a component” includes a plurality of such components, so that acomponent X includes a plurality of components X. It is further notedthat the claims may be drafted to exclude an optional element. As such,this statement is intended to serve as antecedent basis for the use ofexclusive terminology, such as “solely,” “only,” “other than”, and thelike, in connection with any element described herein, and/or therecitation of claim elements or use of “negative” limitations.

The term “and/or” means any one of the items, any combination of theitems, or all of the items with which this term is associated. Thephrases “one or more” and “at least one” are readily understood by oneof skill in the art, particularly when read in context of its usage. Forexample, the phrase can mean one, two, three, four, five, six, ten, 100,or any upper limit approximately 10, 100, or 1000 times higher than arecited lower limit.

The term “about” can refer to a variation of ±5%, ±10%, ±20%, or ±25% ofthe value specified. For example, “about 50” percent can in someembodiments carry a variation from 45 to 55 percent. For integer ranges,the term “about” can include one or two integers greater than and/orless than a recited integer at each end of the range. Unless indicatedotherwise herein, the term “about” is intended to include values, e.g.,weight percentages, proximate to the recited range that are equivalentin terms of the functionality of the individual ingredient, element, thecomposition, or the embodiment. The term about can also modify theend-points of a recited range as discuss above in this paragraph.

As will be understood by the skilled artisan, all numbers, includingthose expressing quantities of ingredients, properties such as molecularweight, reaction conditions, and so forth, are approximations and areunderstood as being optionally modified in all instances by the term“about.” These values can vary depending upon the desired propertiessought to be obtained by those skilled in the art utilizing theteachings of the descriptions herein. It is also understood that suchvalues inherently contain variability necessarily resulting from thestandard deviations found in their respective testing measurements.

As will be understood by one skilled in the art, for any and allpurposes, particularly in terms of providing a written description, allranges recited herein also encompass any and all possible sub-ranges andcombinations of sub-ranges thereof, as well as the individual valuesmaking up the range, particularly integer values. A recited range (e.g.,weight percentages or carbon groups) includes each specific value,integer, decimal, or identity within the range. Any listed range can beeasily recognized as sufficiently describing and enabling the same rangebeing broken down into at least equal halves, thirds, quarters, fifths,or tenths. As a non-limiting example, each range discussed herein can bereadily broken down into a lower third, middle third and upper third,etc. As will also be understood by one skilled in the art, all languagesuch as “up to”, “at least”, “greater than”, “less than”, “more than”,“or more”, and the like, include the number recited and such terms referto ranges that can be subsequently broken down into sub-ranges asdiscussed above. In the same manner, all ratios recited herein alsoinclude all sub-ratios falling within the broader ratio. Accordingly,specific values recited for radicals, substituents, and ranges, are forillustration only; they do not exclude other defined values or othervalues within defined ranges for radicals and substituents.

One skilled in the art will also readily recognize that where membersare grouped together in a common manner, such as in a Markush group, aninvention encompasses not only the entire group listed as a whole, buteach member of the group individually and all possible subgroups of themain group. Additionally, for all purposes, an invention encompasses notonly the main group, but also the main group absent one or more of thegroup members. An invention therefore envisages the explicit exclusionof any one or more of members of a recited group. Accordingly, provisosmay apply to any of the disclosed categories or embodiments whereby anyone or more of the recited elements, species, or embodiments, may beexcluded from such categories or embodiments, for example, for use in anexplicit negative limitation.

The term “contacting” refers to the act of touching, making contact, orof bringing to immediate or close proximity, including at the cellularor molecular level, for example, to bring about a physiologicalreaction, a chemical reaction, or a physical change, e.g., in asolution, in a reaction mixture, in vitro, or in vivo.

For a therapeutic application, an “effective amount” refers to an amounteffective to treat a disease, disorder, and/or condition, or to bringabout a recited effect. For example, an effective amount can be anamount effective to reduce the progression or severity of the conditionor symptoms being treated. Determination of a therapeutically effectiveamount is within the capacity of persons skilled in the art. The term“effective amount” is intended to include an amount of a compositiondescribed herein, or an amount of a combination of peptides describedherein, e.g., that is effective to treat or prevent a disease ordisorder, or to treat the symptoms of the disease or disorder, in ahost. Thus, an “effective amount” generally means an amount thatprovides the desired effect.

Fibroin is a protein derived from the silkworm cocoon (e.g., Bombyxmori). Fibroin includes a heavy chain that is about 350-400 kDa inmolecular weight and a light chain that is about 24-27 kDa in molecularweight, wherein the heavy and light chains are linked together by adisulfide bond. The primary sequences of the heavy and light chains areknown in the art. The fibroin protein chains possess hydrophilic N and Cterminal domains, and alternating blocks of hydrophobic/hydrophilicamino acid sequences allowing for a mixture of steric and electrostaticinteractions with surrounding molecules in solution. At lowconcentration dilutions (1% or less) the fibroin protein molecule isknown to take on an extended protein chain form and not immediatelyaggregate in solution. The fibroin protein is highly miscible withhydrating molecules such as HA, PEG, glycerin, and CMC, has been foundto be highly biocompatible, and integrates or degrades naturally withinthe body through enzymatic action. Native fibroin, or also referred toherein as prior art silk fibroin (PASF), is known in the art and hasbeen described by, for example, Daithankar et al. (Indian J. Biotechnol.2005, 4, 115-121) and International Publication No. WO 2014/145002(Kluge et al.).

The terms “silk-derived protein” (SDP) and “fibroin-derived protein” areused interchangeably herein. These materials are prepared by theprocesses described herein involving heat, pressure, and a highconcentration of a heavy salt solution. Therefore ‘silk-derived’ and‘fibroin-derived’ refer to the starting material of the process thatstructurally modifies the silk fibroin protein to arrive at a proteincomposition (SDP) with the structural, chemical and physical propertiesdescribed herein. The SDP compositions possess enhanced solubility andstability in an aqueous solution. The SDP may be derived from silkwormsilk (e.g., Bombyx mori), spider silk, or genetically engineered silk.

As used herein, the terms “molecular weight” and “average molecularweight” refer to weight average molecular weight determined by standardSodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE)electrophoresis methods undertaken with a NuPAGE™ 4%-12% Bis-Trisprotein gel (ThermoFisher Scientific, Inc.) in combination analysis withImageJ software (National Institutes of Health). ImageJ is used todetermine the relative amount of protein of a given molecular weight ina sample. The software accomplishes this by translating the staining onthe gel (i.e., the amount of protein) into a quantitative signalintensity. The user then compares this signal to a standard (or ladder)consisting of species of known molecular weights. The amount of signalbetween each marker on the ladder is divided by the whole signal. Thecumulative summation of each protein sub-population, also referred toherein as fractions and interchangeably also referred to as fragments,allows the user to determine the median molecular weight, which isreferred to herein as the average molecular weight. In practice,electrophoresis gels are stained, and then scanned into greyscaleimages, which are converted into histograms using ImageJ. Total pixelintensity within each gel lane is quantified by ImageJ (i.e., total areaunder the histogram), and subsequently fractionated into populationsdemarcated by protein molecular weight standards also stained on thegel. The histogram pixel area between any two molecular weight standardsis divided by the total histogram area of the protein, thereby providingthe fraction of total protein that falls within these molecular weights.Analysis by other methods may provide different values that account forcertain peptides that are not accounted for by SDS-PAGE methods. Forexample, HPLC can be used to analyze the average molecular weights,which method provides values that are typically about 10-30%, lower thandetermined by SDS-PAGE (increasing differences as molecular weightsdecrease).

Preparation of SDP Compositions

SDP compositions described herein can possess enhanced stabilitycompared to native fibroin in aqueous solutions. The enhanced stabilityachieved by the SDP compositions provided herein, which is also referredherein as a SDP, allow the material to remain in solution significantlylonger than the native/PASF proteins (referred to herein as PASF).Enhanced stability of the SDP materials provided herein also allow forthe preparation of SDP solutions of high concentration withoutaggregation, precipitation, or gelation. In commercial applications suchas eye drops or applications requiring protein to be soluble insolution, enhanced stability can provide suitably lengthy shelf life andincreased quality of the product by reducing protein aggregation.Potential aggregation of protein in solution can negatively impact aproduct's desired performance for a particular application. This isespecially true for eye drop formulations given that aggregates couldcause abrasive damage to the ocular surface. The ability to concentratethe SDP to high constitutions in solution (over 50% w/v or >500 mg/mL)is significantly advantageous for inventorying a useful working solutionthat can be used as-is or diluted for any number of applications.Examples of such applications are the use of SDP as an ingredient inophthalmic formulations, such as those provided herein, as a proteinsupplement or additive.

Transforming the primary amino acid sequences of the native fibroinprotein into the SDP material may enhance its stability in aqueoussolutions by decreasing the susceptibility of the molecules toaggregate. Aggregation eventually leads to gel formation. In thetransformation of the native fibroin, both serine and cysteine aminoacids are cleaved in the presence of high heat and dehydratingconditions. Similarly, Patchornik et al. (J. Am. Chem. Soc. 1964, 86,1206) demonstrated that a dehydroalanine (DHA) intermediate is formedfrom serine and cysteine in solution. The amino acid degradation isfurther driven when in the presence of a strong dehydrating solventsystem, such as the 50-55% w/v LiBr solution as described herein, inwhich a hydride shift takes place to induce removal of water. Thedegradation reaction can take place in the presence of hydroxide ions(e.g., pH 7.5 to pH 11), which further drives cleavage of the DHAintermediate. This cleavage forms an amide, a pyruvoyl peptide, andLiBr. One viable chemical mechanism is outlined in Scheme 1 for a serineamino acid, which scheme is also applicable for cysteine amino acids.Chemical alteration of the serine and cysteine amino acids of the PASFprotein into a DHA intermediate with further hydrolytic cleavage leadsto enhanced solution stability of the SDP products.

Degradation is driven by the production of a DHA intermediate that isformed from a hydride shift reaction in the presence of a dehydratinghigh salt concentration environment. Degradation of DHA is thenaccomplished through an SN₂ reaction within the basic solventenvironment.

This cleavage reaction discussed above can significantly affectmacromolecular properties of the resulting peptides, which results in anaqueous solution of stabilized SDP material. The initial proteinaggregation of fibroin is believed to be instigated by interactions ofthe native fibroin heavy and light chains at the cysteine amino acids asdescribed by Greying et al. (Biomacromolecules 2012, 13(3): 676-682).The cysteine amino acids within the fibroin light and heavy proteinchains interact with one another through disulfide linkages. Thesedisulfide bridges participate in fibroin protein aggregation and gelnetwork flocculation. Without the native fibroin light chain present,the proteins are significantly less susceptible to aggregation.Therefore, the process described herein can effectively reduce\ thenative fibroin light chain's ability to form disulfide bonds by reducingcysteine content and thus reducing or eliminating disulfide bond-formingcapability. Through this mechanism, the transformative process describedherein functionally stabilizes the resulting SDP in solution by reducingor eliminating the ability to form cysteine-derived aggregations.

In addition to aggregation-inducing disulfide bridges, thesusceptibility of the silk fibroin to further aggregate into flocculatedstructure is also driven by the protein's amino acid chemistry asdescribed by Mayen et al. (Biophysical Chemistry 2015, 197:10-17).Molecular modeling of silk fibroin serine, alanine, and glycine aminoacid sequences have shown that the presence of serine enhances initialprotein-to-protein interaction through a greater propensity to createhydrogen bonding between adjacent fibroin protein chain moieties. Themodels demonstrate that reduced serine and increased alanine and glycinedecrease the initial propensity for protein aggregation. The molecularmodeling observations indicate that by altering the native amino acidchemistry of the fibroin protein a material could be generated thatwould have higher stability in aqueous solution.

One strategy to accomplish enhanced stability is to eliminate chargedfunctional groups, such as hydroxyls, from the protein. Due to therelatively high electronegativity of hydroxyl groups, this chemistry candrive both hydrogen bonding with available hydrogen atoms andnon-specific charge interactions with positively charged amino acidgroups. Almost 12% of the native fibroin protein's content is composedof serine, which bears a hydroxyl functional group. Therefore, byreducing the availability of hydroxyl groups that facilitate hydrogenbonding, the overall protein stability in solution may be enhanced. Theprocess described herein effectively reduces the amount of serinecontent and increases the relative alanine and glycine content, whicheliminates the number of available hydroxyl groups available to createhydrogen bonds. Through this mechanism the process described hereinfunctionally stabilizes the resulting SDP in solution extended periodsof time (e.g., at least several [6-8] months, and/or for more than 1.5years; extended studies are ongoing, indicating that stability may bemaintained for more than 2 years, or more than 3 years).

In addition to the reduction of cysteine and serine moieties, solventcharge interaction is important for stabilizing a protein solution.After initial protein flocculation, the gelation process is believed tocontinue to drive closer associations among the native fibroin heavychains, which leads to both intra- and inter-molecular beta-sheetformation among hydrophobic blocks of the heavy chains. Once significantbeta-sheet formation occurs, the fibroin solution transitions to a gel.As the solution transitions to a gel, and the fibroin becomes insolubleand is no longer useful as a solution-based product. To preventgelation, the pH of the SDP solution can be raised to high alkalinity toenhance stability, for example over a pH of 7.5. As a result, theincreased pH produces additional free hydroxyl groups that form avalence shield around the SDP molecules in solution. The formed valenceshield acts to produce a zeta potential that stabilizes the protein byreducing protein-protein interactions derived from hydrogen bonding ornon-specific charged and/or hydrophobic interactions. Thefibroin-transformation process functionally stabilizes processed SDP insolution through this mechanism and others. The SDP can be derived fromBombyx mori silkworm fibroin or other fibroin from the Bombyx genus orother silk proteins.

SDP material can be prepared by the following process.

1. Silk cocoons are prepared by removing pupae material and pre-rinsingin warm water.

2. Native fibroin protein fibers are extracted from the gum-like sericinproteins by washing the cocoons in water at high water temperature,typically 95° C. or more, at alkaline pH.

3. The extracted fibroin fibers are dried and then dissolved using asolvent system that neutralizes hydrogen bonding between thebeta-sheets; a 54% LiBr aqueous solution of 20% w/v silk fibroin proteinis effective for this neutralization step.

4. The fibroin protein dissolved in LiBr solution is processed in anautoclave environment (˜121° C. [˜250° F.], at ˜15-17 PSI pressure, forapproximately 30 minutes at temperature).

5. The heat-processed fibroin protein and LiBr solution are thendialyzed to remove lithium and bromide ions from the solution. At thispoint in the process the material has been chemically transformed toSDP.

6. The dialyzed SDP is then filtered to remove any non-dissolvedaggregates and contaminating bioburden.

The SDP solution is produced using a distinctly different process thanthe process used for current silk fibroin solution production. Notably,the autoclaving of the silk fibroin protein while it is combined withLiBr in solution initiates chemical transitions to produce thestabilized SDP material. The fibroin protein is dissolved in LiBrsolution, which neutralizes hydrogen bonding and electrostaticinteractions of the solubilized native fibroin protein. This leaves theprotein without specific secondary structure confirmations in solution.As a result, the thermodynamic energy required to hydrolyze covalentbonding within the fibroin protein chain is at its lowest energyrequirements to initiate hydrolytic cleavage.

In one embodiment, the temperature is set to 121° C. for 30 minutes at15-17 PSI autoclave conditions. However, in various embodiments, theprocessing conditions may be modified to stabilize the SDP material tovarying degrees. In other embodiments, additional protein solubilizationagents can be used in the process, including other or additional halidesalts such as calcium chloride and sodium thiocyanate, organic agentssuch as urea, guanidine hydrochloride, and1,1,1,3,3,3-hexafluoroisopropanol, additional strong ionic liquidsolution additives such as calcium nitrate and1-butyl-3-methylimidazolium chloride, or a combination thereof.

SDP Compositions

Protein composition described herein can be derived from silk fibroinand possess enhanced solubility and stability in aqueous solutions. Thecompositions can be used to treat and reduce inflammation. In oneembodiment, the SDP and/or fractions thereof have primary amino acidsequences that differ from native fibroin by at least 4% (via summationof the absolute values of the differences) with respect to the combinedamino acid content of serine, glycine, and alanine. A plurality of theprotein fragments of SDP can terminate in amide (—C(═O)NH₂) groups. SDPcan have a serine content that is reduced by greater than 40% comparedto native fibroin, wherein the serine content is at least about 5%. Thecysteine disulfide bonds between the fibroin heavy and fibroin lightprotein chains of fibroin may be reduced or eliminated. The SDPcompositions provided herein possess enhanced stability in an aqueoussolution. In certain embodiments, at least 75 percent of the proteinfragments have a molecular weight of less than about 60 kDa and act asan anti-inflammatory that also promotes cell migration and proliferationin the tissue to close the wound. The composition may comprise less than8.5% serine amino acid residues. In some embodiments, the averagemolecular weight of the SDP is less than 55 kDa.

In some cases, protein compositions provided herein are prepared by aprocess comprising heating an aqueous fibroin solution at an elevatedpressure. The aqueous fibroin solution includes lithium bromide at aconcentration of at least 8M. The aqueous fibroin solution is heated toat least about 105° C. (221° F.) under a pressure of at least about 10PSI for at least about 20 minutes, to provide the protein composition.As a result of these processing conditions, the polypeptides of theprotein composition comprise less than 8.5% serine amino acid residues,and a plurality of the protein fragments terminate in amide (C(═O)NH₂)groups.

In some cases, protein compositions provided herein are prepared by aprocess comprising heating an aqueous fibroin solution at an elevatedpressure, wherein the aqueous fibroin solution comprises lithium bromideat a concentration of 9-10M, and wherein the aqueous fibroin solution isheated to a temperature in the range of about 115° C. (239° F.) to about125° C. (257° F.), under a pressure of about 15 PSI to about 20 PSI forat least about 20 minutes; to provide the protein composition. Theprotein composition can include less than 6.5% serine amino acidresidues.

SDP compositions provided herein can possess enhanced stability inaqueous solution, wherein: the primary amino acid sequences of the SDPcomposition differs from native fibroin by at least 4% with respect tothe combined (absolute value) difference in serine, glycine, and alaninecontent (SDP vs. PASF); cysteine disulfide bonds between the fibroinheavy and fibroin light protein chains are reduced or eliminated; andthe composition has a serine content that is reduced by greater than 25%compared to native fibroin protein. The average molecular weight of theSDP composition can be less than about 60 kDa and greater than about 2kDa, or greater than about 10 kDa, as determined by the MWCO of thedialyzing membrane and SDS-PAGE analysis.

In some cases, SDP compositions provided herein possess enhancedstability in aqueous solution, wherein: the primary amino acid sequencesof the SDP composition differs from native fibroin by at least 6% withrespect to the combined difference in serine, glycine, and alaninecontent; cysteine disulfide bonds between the fibroin heavy and fibroinlight protein chains are reduced or eliminated; and the composition hasa serine content that is reduced by greater than 40% compared to nativefibroin protein. The average molecular weight of the SDP composition canbe less than about 55 kDa and greater than about 10 kDa, as determinedby the MWCO of the dialyzing membrane and SDS-PAGE analysis.

In some cases, SDP compositions provided herein possess enhancedstability in aqueous solutions, wherein: the primary amino acidsequences of the SDP composition is modified from native silk fibroin;cysteine disulfide bonds between the fibroin heavy and fibroin lightprotein chains are reduced or eliminated; the average molecular weightof the SDP composition is less than about 60 kDa and greater than about10 kDa; and a 5% w/w aqueous solution of the SDP composition maintainsan optical absorbance at 550 nm of less than 0.25 for at least two hoursafter five seconds of sonication.

In some cases, SDP compositions provided herein possess enhancedstability in aqueous solutions, wherein: the primary amino acidsequences of the SDP composition is modified from native silk fibroinsuch that they differ from native fibroin by at least 5% with respect tothe combined (absolute value) difference in serine, glycine, and alaninecontent. In some embodiments, the difference of is at least 6%, 8%, 10%,12% or 14% compared to native fibroin. Cysteine disulfide bonds betweenthe fibroin heavy and fibroin light protein chains are reduced oreliminated; the average molecular weight of the SDP composition is lessthan about 60 kDa and greater than about 15 kDa; and the SDP compositionmaintains an optical absorbance at 550 nm of less than 0.2 for at leasttwo hours after five seconds of sonication.

In some cases, SDP compositions provided herein can be isolated and/orpurified as a dry powder or film, for example, by dialysis and/orfiltration. Alternatively, SDP compositions provided herein can beisolated and/or purified as a stable aqueous solution, which can bemodified for use as a therapeutic formulation, such as an ophthalmicformulation.

In various embodiments, the amino acid compositions of the SDP found inprotein compositions provided herein can differ from the amino acidcomposition of native fibroin by at least 4%, by at least 4.5%, by atleast 5%, or by at least 5.5%, or by at least 6%, with respect to thecontent of serine, glycine, and alanine combined.

In some cases, protein compositions described herein have a serinecontent that is reduced by greater than 25%, by greater than 30%, bygreater than 35%, by greater than 40%, or by greater than 45%, comparedto the serine content of native fibroin protein.

The average molecular weight of SDP compositions provided herein can beless than about 80 kDa, less than about 70 kDa, less than about 60 kDa,or less than about 55 kDa, or the composition has an average molecularweight of about 50-60 kDa, or about 51-55 kDa. In various embodiments,the average molecular weight of the SDP composition can be greater thanabout 2 kDa, greater than about 10 kDa, greater than about 15 kDa,greater than about 20 kDa, greater than about 25 kDa, greater than about30 kDa, greater than about 35 kDa, greater than about 40 kDa, or greaterthan about 50 kDa. Accordingly, the (weight average) average molecularweight of SDP compositions provided herein can be about 5 kDa to about80 kDa, about 10 kDa to about 65 kDa, about 15 kDa to about 60 kDa,about 15 kDa to about 60 kDa, about 20 kDa to about 65 kDa, about 20 kDato about 55 kDa. In various embodiments, the average molecular weight ofthe SDP composition is about 45 kDa to about 65 kDa, about 45 kDa toabout 60 kDa, about 50 kDa to about 65 kDa, or about 50 kDa to about 60kDa.

The SDP protein compositions can be soluble in water at 40% w/w withoutany precipitation observable by ocular inspection.

In some embodiments, protein compositions provided herein comprise lessthan 8% serine amino acid residues. In some cases, protein compositionsprovided herein comprise less than 7.5% serine amino acid residues, lessthan 7% serine amino acid residues, less than 6.5% serine amino acidresidues, or less than 6% serine amino acid residues. The serine contentof the peptide compositions is generally at least about 4%, or at leastabout 5%, or about 4-5%.

In some embodiments, protein compositions provided herein comprisegreater than 46.5% glycine amino acids, relative to the total amino acidcontent of the protein composition. In some cases, protein compositionsprovided herein comprise greater than 47% glycine amino acids, greaterthan 47.5% glycine amino acids, or greater than 48% glycine amino acids.

In some embodiments, protein compositions provided herein comprisegreater than 30% alanine amino acids, relative to the total amino acidcontent of the protein composition. In some cases, protein compositionsprovided herein comprise greater than 30.5% alanine, greater than 31%alanine, or greater than 31.5% alanine.

In some embodiments, protein compositions provided herein can completelyre-dissolve after being dried to a thin film. In various embodiments,protein compositions provided herein can lack beta-sheet proteinstructure in aqueous solution. The protein composition can maintain anoptical absorbance in aqueous solution of less than 0.25 at 550 nm afterat least five seconds of sonication.

In some embodiments, protein compositions provided herein can be incombination with water. In some cases, protein compositions providedherein can completely dissolve in water at a concentration of 10% w/w,or even greater concentrations such as 15% w/w, 20% w/w, 25% w/w, 30%w/w, 35% w/w, or 40% w/w. In some embodiments, protein compositionsprovided herein can be isolated and purified, for example, by dialysis,filtration, or a combination thereof.

In various embodiments, protein compositions provided herein can enhancethe spreading of an aqueous solution comprising the protein compositionand ophthalmic formulation components, for example, compared to thespreading of a corresponding composition that does not include theprotein composition. This enhanced spreading can result in an increasein surface area of the aqueous solution by greater than twofold, orgreater than threefold.

In various embodiments, the SDP protein compositions do not form a gelat concentrations up to 20% w/v, up to 30% w/v, or up to 40% w/v inwater. In some embodiments, SDP compositions provided herein can haveglycine-alanine-glycine-alanine (GAGA) (SEQ ID NO: 1) segments of aminoacids that comprise at least about 47.5% of the amino acids of the SDPcomposition. In some cases, SDP compositions provided herein can alsohave GAGA (SEQ ID NO: 1) segments of amino acids that comprise at leastabout 48%, at least about 48.5%, at least about 49%, at least about49.5%, or at least about 50%, of the amino acids of the proteincomposition.

In various embodiments, SDP compositions provided herein can haveglycine-alanine (GA) segments of amino acids that comprise at leastabout 59% of the amino acids of the SDP composition. In some cases, SDPcompositions provided herein can also have GA segments of amino acidsthat comprise at least about 59.5%, at least about 60%, at least about60.5%, at least about 61%, or at least about 61.5%, of the amino acidsof the protein composition.

Protein compositions provided herein can be prepared by a processcomprising heating an aqueous fibroin solution at an elevated pressure,wherein the aqueous fibroin solution comprises lithium bromide at aconcentration of at least 8M, and wherein the aqueous fibroin solutionis heated to at least about 105° C. (221° F.) under a pressure of atleast about 10 PSI for at least about 20 minutes; to provide the proteincomposition, wherein the protein composition comprises less than 8.5%serine amino acid residues. Therefore, methods of preparing a SDPcomposition are also provided herein. Methods of preparing a SDPcomposition provided herein can include one or more of the process stepsdescribed herein.

In some cases, methods of preparing provided herein can use lithiumbromide having a concentration between about 8.0M and about 11M. In someembodiments, the concentration of lithium bromide is about 9M to about10M, or about 9.5M to about 10M.

In some embodiments, the aqueous fibroin solution that contains lithiumbromide is heated to at least about 107° C. (225° F.), at least about110° C. (230° F.), at least about 113° C. (235° F.), at least about 115°C. (239° F.), or at least about 120° C. (248° F.).

In some embodiments, the aqueous fibroin solution that contains lithiumbromide is heated under a pressure of at least about 12 PSI, at leastabout 14 PSI, at least about 15 PSI, or at least about 16 PSI, up toabout 18 PSI, or up to about 20 PSI.

In some embodiments, the aqueous fibroin solution that contains lithiumbromide is heated for at least about 20 minutes, at least about 30minutes, at least about 45 minutes, or at least about 1 hour, up toseveral (e.g., 12-24) hours.

In some embodiments, the protein composition can be dissolved in waterat 40% w/w without observable precipitation.

In some embodiments, the fibroin has been separated from sericin.

In some embodiments, lithium bromide has been removed from the proteincomposition to provide a purified protein composition. In variousembodiments, the protein composition has been isolated and purified, forexample, by dialysis, filtration, or a combination thereof.

In additional embodiments, the protein composition has properties asdescribed above, and amino acid compositions as described aboveregarding serine, glycine, and alanine content.

In various embodiments, the protein composition re-dissolves afterdrying as a thin film, a property not found with native fibroin. Theprotein composition can lack beta-sheet protein structure in solution.The protein composition can maintain an optical absorbance in solutionof less than 0.25 at 550 nm after at least five seconds of sonication.

In one specific embodiment, the invention provides a protein compositionprepared by a process comprising heating an aqueous fibroin solution atan elevated pressure, wherein the aqueous fibroin solution compriseslithium bromide at a concentration of 9-10M, and wherein the aqueousfibroin solution is heated to a temperature in the range of about 115°C. (239° F.) to about 125° C. (257° F.), under a pressure of about 15PSI to about 20 PSI for at least about 30 minutes; to provide theprotein composition, wherein the protein composition comprises less than6.5% serine amino acid residues. and the protein composition has anaqueous viscosity of less than 10 cP as a 15% w/w solution in water.

SDP compositions are chemically distinct from native silk fibroinprotein as a result of the preparation process, resulting in changes inamino acid content and the formation of terminal amide groups. Theresulting SDP has enhanced solubility and stability in aqueous solution.The SDP can be used in a method for forming, for example, ophthalmicformulations with a protein composition described herein, for example,an aqueous solution of the protein composition. The solution can includeabout 0.01% to about 92% w/v SDP. The solution can be about 8% to about99.9% w/v water.

In some embodiments, processes are provided that induces hydrolysis,amino acid degradation, or a combination thereof, of fibroin proteinsuch that the average molecular weight of the protein is reduced fromabout 100-200 kDa for silk fibroin produced using prior art methods toabout 30-90 kDa, or about 30-50 kDa, for the SDP material describedherein. The resulting polypeptides can be a random assortment ofpeptides of various molecular weights averaging to the ranges recitedherein.

In addition, the amino acid chemistry can be altered by reducingcysteine content to non-detectable levels by standard assay procedures.For example, the serine content can be reduced by over 50% from thelevels found in the native fibroin, which can result in increases ofoverall alanine and glycine content by 5% (relative amino acid content),as determined by standard assay procedures. The SDP material can have aserine content of less than about 8% relative amino acid content, or aserine amino acid content of less than about 6% relative amino acidcontent. The SDP material can have a glycine content above about 46.5%,and/or an alanine content above about 30% or above about 30.5%. The SDPmaterial can have no detectable cysteine content, for example, asdetermined by HPLC analysis of the hydrolyzed polypeptide of the proteincomposition. The SDP material can form 90% less, 95% less, or 98% lessbeta-sheet secondary protein structures as compared to native silkfibroin protein, for example, as determined by the FTIR analysis.

Stability Evaluations. The stability of a protein solution can beevaluated a number of different ways. One suitable evaluation is theLawrence Stability Test described below in Example 1 below. Anothersuitable evaluation is the application of sonication to a proteinsolution, followed by optical absorbance analysis to confirm continuedoptical clarity (and lack of aggregation, beta-sheet formation, and/orgelation). Standard sonication, or alternatively ultrasonication (soundfrequencies greater than 20 kHz), can be used to test the stability ofan SDP solution. Solutions of SDP are stable after subjecting tosonication. The SDP composition maintains an optical absorbance at 550nm of less than 0.25 for at least two hours after five seconds ofsonication. For example, a 5% w/w solution of the protein compositioncan maintain an optical absorbance of less than 0.1 at 550 nm after fiveseconds of sonication at ˜20 kHz, the standard conditions used for thesonication described herein. In various embodiments, SDP compositionaqueous solutions do not gel upon sonication at concentrations of up to10% w/w. In further embodiments, SDP composition aqueous solutions donot gel upon ultrasonication at concentrations of up to 15% w/w, up to20% w/w, up to 25% w/w, up to 30% w/w, up to 35% w/w, or up to 40% w/w.

Low viscosity. As a result of its preparation process and the resultingchanges in the chemical structures of its peptide chains, SDP has alower viscosity than native silk fibroin (PASF). As a 5% w/w solution inwater (at 25.6° C.), native silk fibroin has a viscosity of about 5.8cP, whereas under the same conditions, SDP has a viscosity of about 1.8cP, and SDP-4 has a viscosity of about 2.7 cP. SDP maintains a lowviscosity compared to PASF at higher concentrations as well. The SDPcomposition can have an aqueous viscosity of less than 5 cP, or lessthan 4 cP, as a 10% w/w solution in water. In various embodiments, SDPremains in solution up to a viscosity of at least 9.8 cP. SDP also hasan aqueous viscosity of less than 10 cP as a 15% w/w solution in water.SDP can also have an aqueous viscosity of less than 10 cP as a 24% w/wsolution in water.

The process described herein provides a protein composition where thefibroin light chain protein is not discernable after processing, as wellwhen the sample is run using standard Sodium Dodecyl SulfatePolyacrylamide Gel Electrophoresis (SDS-PAGE) electrophoresis methodsundertaken with a NuPAGE™ 4%-12% Bis-Tris protein gel (ThermoFisherScientific, Inc.). For example, in one embodiment, the SDP material canhave the fibroin light chain over 50% removed when compared to nativesilk fibroin protein. Furthermore, the resulting SDP material formsminimal to no beta-sheet protein secondary structure post-processing,while silk fibroin solution produced using prior art methods formssignificant amounts of beta-sheet secondary structure. In oneembodiment, the SDP material can be prepared by processing silk fibroinfibers under autoclave or autoclave-like conditions (i.e., approximately120° C. and 14-18 PSI) in the presence of a 40-60% w/v lithium bromide(LiBr) solution.

SDP Composition Fractions

Silk Technologies, Ltd. has developed the silk-derived protein (SDP)product that can be readily incorporated into ophthalmic productformulations for reducing inflammation and enhancing the wound healingprocess. The SDP product can be separated into smaller protein fractionsor sub-populations based on molecular weight to enhance theanti-inflammatory and wound healing properties. SDP proteinsub-populations, also referred to as fractions or fragments, can beseparated by any suitable and effective method, for example, by sizeexclusion chromatography or membrane dialysis. For example, thefractions can be separated in to 2-4 different groups based ondecreasing average molecular weights. Example 6 describes one method forpreparing four different fractions that have the same overall amino acidcontent and terminal amide content but different average molecularweights. It was surprisingly discovered that the different fractionsalso possess different biological properties, for example, for reducinginflammation in the body and in various tissues as a result ofdifferences in cellular uptake of the different fractions.

This disclosure therefore provides methods of reducing inflammationand/or enhancing wound healing using SDP, including low averagemolecular weight fractions of SDP. Also described are compositions forreducing inflammation in the treatment of ocular conditions, such as,but not limited to, dry eye disease, and/or injury, including cornealwounds. The treatments can include the administration of a formulationthat includes SDP, or a low molecular weight SDP sub-population. Incertain embodiments, the invention provides methods for treating adisease state and/or wound comprising administering to a subject in needthereof a composition comprising low molecular weight SDP (e.g., SDP-3or SDP-4).

The methods can include applying a composition of SDP fractions todiseased or injured tissue. The protein fractions can have primary aminoacid sequences that differ (via summation of absolute value differences)from native fibroin by at least 4% with respect to the combined aminoacid content of serine, glycine, and alanine. A plurality of the proteinfragments can terminate in amide (—C(═O)NH₂) groups. Compositionsprovided herein may have a serine content that is reduced by greaterthan 40% compared to native fibroin, wherein the serine content is atleast about 5%. The cysteine disulfide bonds between the fibroin heavyand fibroin light protein chains of fibroin may be reduced oreliminated. In some embodiments, at least 75 percent of the proteinfragments have a molecular weight of less than about 100 kDa. Suchcompositions reduce inflammation, and promote cell migration and/orproliferation in the tissue to treat the disease state and/or enhanceclosure of the wound. The SDP compositions possess enhanced solubilityand stability in an aqueous solution.

SDP composition fractions can have an average molecular weight betweenabout 2 kDa and 60 kDa. In one embodiment, a low molecular weightfraction having an average molecular weight of 25-38 kDa, of 32-35 kDa,or about 34 kDa±5%, is isolated, which fraction is referred to herein asSDP-4.

In some embodiments, at least 60 percent of the protein fragments have amolecular weight of less than about 60 kDa, or less than about 55 kDa,to promote cell migration and proliferation in the tissue to close thewound. In another embodiment, at least 90 percent of the proteinfragments have a molecular weight of less than about 100 kDa and promotecell migration and proliferation in the tissue to close the wound.

In some embodiments, at least 80 percent of the protein fragments have amolecular weight between about 10 kDa and 85 kDa. In some embodiments,at least 50 percent of the protein fragments have a molecular weightbetween about 20 kDa and 60 kDa. In some embodiments, at least 85percent of the protein fragments have a molecular weight of greater thanabout 10 kDa. In some embodiments, at least 90 percent of the proteinfragments have a molecular weight of greater than about 5 kDa.

In certain embodiments, the invention provides an SDP compositioncomprising low molecular weight SDP and a pharmaceutically acceptablecarrier. The low molecular weight SDP can have an average molecularweight of less than 60 kDa. In some embodiments, the low molecularweight SDP is less than 40 kDa and the fraction reduces inflammationand/or enhances cell migration and/or proliferation.

In one embodiment, the low molecular weight SDP, for example, SDP-4, is10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater of the total SDPin a composition. In some embodiments, the composition does not comprisehigh molecular weight SDP, for example, the sample has an averagemolecular weight of less than about 35 kDa.

In one embodiment, the SDP-4 fraction has an average molecular weight of33-35 kDa, as determined by SDS-PAGE/ImageJ analysis, as previouslydescribed above, and a pH 8.1-8.3, an osmolarity of about 23 mOsm, and aviscosity of about 1.5-3 cP at 25° C., each as a 50 mg/mL solution inwater.

Various compositions can be prepared to include low molecular weightprotein fragments or high molecular weight protein fragments orcombinations thereof. Low molecular weight protein fragments can reduceinflammation and/or enhance cell migration and/or proliferation on adiseased tissue surface and/or wound. Low molecular weight proteinfragments are also useful in treating inflamed tissue surfaces due to anactive disease state and/or the presence of a wound or wounds. In somecases, it may be useful to apply a composition of low molecular weightprotein fragments to enhance the wound healing process. These cases mayinclude wounds acquired on the battlefield during war, surgical woundsof a person who desires faster healing, for example, of an infection orfor pain relief. The wound healing process is enhanced by increasingcell numbers, reducing inflammatory molecules, such as MMP-9, and/orincreasing epithelial cell proliferation.

High molecular weight protein fragments may increase cell adhesion tothe basement membrane or aid in basement membrane formation. In somecases, it may be useful to apply a composition of high molecular weightprotein fragments for chronic wounds or wounds that fester or woundsthat have difficulty healing up, such as diabetic ulcers or skin burns.Whereas low molecular weight protein fragments may be involved in woundclosure rate, high molecular weight protein fragments may be involved inwound closure quality. In some cases, it may be used to apply acomposition of carefully selected amounts of low molecular weightprotein fragments and high molecular weight protein fragments foroptimal wound healing rate and quality. The wound healing process isenhanced by increasing structural proteins, such focal adhesion kinases(FAK) and/or tight junctions between cells, such as zonula occluden(ZO-1) structures.

Low average molecular weight fractions such as SDP-4 possess certainproperties making the fraction distinct from SDP and higher molecularweight fractions. For example, SDP cellular uptake is dependent onmolecular weight of the peptide chains. SDP peptide molecules smallerthan about 60 kDa in size are readily absorbed by cells in culture, andmore specifically human corneal limbal epithelial (hCLE) cells. SDPmolecules larger than about 60 kDa in size are mostly excluded frombeing absorbed by the cell cultures. It is also important to note thatSDP molecules do not co-localize with lysosomal-associated membraneprotein 1 (LAMP-1), which is a marker for the lysosomal endocytoticdegradation pathway. As a result, the SDP molecules appear to associatewith a non-specified cellular membrane receptor, in which molecules ofless than about 60 kDa are then absorbed by the hCLE cells. Moreimportantly, because the SDP molecules are not absorbed through thelysosomal degradation pathway they are bioavailable and able to elicitbiological activity.

SDP Formulations

The SDP compositions and sub-fractions described herein can beformulated with water and/or a pharmaceutical carrier. Thepharmaceutical carrier can be, for example, phosphate buffered saline, afilm, a fiber, a foam, a hydrogel, a protein or polymer matrix, athree-dimensional scaffold, a microparticle, a nanoparticle, a polymer,or a mat. In some embodiments, the protein fragments may be attached toa substrate such as a corneal transplant, a wound dressing, a contactlens, a tissue, a tissue-graft, or a degradable material. In a specificembodiment, the carrier is phosphate buffered saline, for example, in anocular formulation.

In some embodiments, ophthalmic compositions are provided for thetreatment of dry eye syndrome in a human or mammal. Compositionsprovided herein can be an aqueous solution that includes an amount ofSDP effective for treating dry eye syndrome. For example, the effectiveamount of the SDP in the aqueous solution can be about 0.01% by weightto about 80% by weight SDP. In other embodiments, the aqueous solutioncan include SDP at about 0.1% by weight to about 10% by weight, or about0.5% by weight to about 2% by weight. In certain specific embodiments,the ophthalmic composition can include about 0.05% w/v SDP, about 0.1%w/v SDP, about 0.2% w/v SDP, about 0.25% w/v SDP, about 0.5% w/v SDP,about 0.75% w/v SDP, about 1% w/v SDP, about 1.5% w/v SDP, about 2% w/vSDP, about 2.5% w/v SDP, about 5% w/v SDP, about 8% w/v SDP, or about10% w/v SDP.

In various embodiments, the ophthalmic formulation can includeadditional components in the aqueous solution, such as a demulcentagent, a buffering agent, and/or a stabilizing agent. The demulcentagent can be, for example, hyaluronic acid (HA), hydroxyethyl cellulose,hydroxypropyl methylcellulose, dextran, gelatin, a polyol, carboxymethylcellulose (CMC), polyethylene glycol, propylene glycol (PG),hypromellose, glycerin, polysorbate 80, polyvinyl alcohol, or povidone.The demulcent agent can be present, for example, at about 0.01% byweight to about 10% by weight, or at about 0.2% by weight to about 2% byweight. In one specific embodiment, the demulcent agent is HA. Invarious embodiments, the HA can be present at about 0.2% by weight ofthe formulation.

The buffering or stabilizing agent of an ophthalmic formulation can bephosphate buffered saline, borate buffered saline, citrate buffersaline, sodium chloride, calcium chloride, magnesium chloride, potassiumchloride, sodium bicarbonate, zinc chloride, hydrochloric acid, sodiumhydroxide, edetate disodium, or a combination thereof.

An ophthalmic formulation can further include an effective amount of anantimicrobial preservative. The antimicrobial preservative can be, forexample, sodium perborate, polyquaterium-1 (e.g., Polyquad®preservative), benzalkonium (BAK) chloride, sodium chlorite,brimonidine, brimonidine purite, polexitonium, or a combination thereof.

An ophthalmic formulation can also include an effective amount of avasoconstrictor, an anti-histamine, or a combination thereof. Thevasoconstrictor or antihistamine can be naphazoline hydrochloride,ephedrine hydrochloride, phenylephrine hydrochloride, tetrahydrozolinehydrochloride, pheniramine maleate, or a combination thereof.

In one embodiment, an ophthalmic formulation can include an effectiveamount of SDP as described herein in combination with water and one ormore ophthalmic components. The ophthalmic components can be, forexample, a) polyvinyl alcohol; b) PEG and hyaluronic acid; c) PEG andpropylene glycol, d) CMC and glycerin; e) propylene glycol and glycerin;f) glycerin, hypromellose, and PEG; or a combination of any one or moreof the preceding components. The ophthalmic formulation can include oneor more inactive ingredients such as HP-guar, borate, calcium chloride,magnesium chloride, potassium chloride, zinc chloride, and the like. Theophthalmic formulation can also include one or more ophthalmicpreservatives such as sodium chlorite (Purite® preservative (NaClO₂),polyquad, BAK, EDTA, sorbic acid, benzyl alcohol, and the like.Ophthalmic components, inactive ingredients, and preservatives can beincluded at about 0.1% to about 5% w/v, such as about 0.25%, 0.3%, 0.4%,0.5%, 1%, 2%, 2.5%, or 5%, or a range in between any two of theaforementioned values.

Ophthalmic formulations for the treatment of ophthalmic disorders in ahuman or mammal can be prepared, wherein the ophthalmic formulationcomprises water and an effective amount of the SDP as described above.The ophthalmic composition can be used as an eye treatment in a human ormammal, where the ophthalmic composition comprises water, one or more ofa buffering agent and stabilizing agent, and an effective amount of theSDP or a sub-fraction thereof.

The SDP is highly stable in water, where shelf life solution stabilityis more than twice that of native silk fibroin in solution. For example,the SDP is highly stable in water, where shelf life solution stabilityis more than 10 times greater compared to native silk fibroin insolution. The SDP material, when in an aqueous solution, does not gelupon sonication of the solution at a 5% (50 mg/mL) concentration. Inother embodiments, the SDP material, when in an aqueous solution, doesnot gel upon sonication of the solution at a 10% (100 mg/mL)concentration.

Therapeutic Methods

The invention provides for the use of SDP in formulations to reduceinflammation, for example, inflammation on or in the human cornea. Suchreduction in inflammation has been demonstrated in both in vitro and invivo experimental models. Specifically, work was undertaken to show thatSDP works to reduce inflammation in human corneal models by inhibitingNF-κB-associated cell signaling pathways (see FIGS. 1 and 2), which is aknown driver of inflammation in the body, in which one specific exampleis dry eye disease. It was found that inhibition of these pathwaysultimately led to reduced genetic expression and tissue residence ofMMP-9, which is a known driver of dry eye and ocular inflammation (seeFIG. 4). Although the studies listed here are specific to cornealinflammation, the biological processes affected are also presentthroughout the various tissues of the body. As a result, the workdisclosed herein regarding the cornea can be extended to other tissuesystems containing an epithelial surface, in which one such example isskin.

The invention thus provides methods for reducing inflammation and fortreating wounds, including corneal wounds, comprising the administrationof SDP to the site of interest. The methods can include administering aformulation comprising a composition of silk-derived protein (SDP), ormolecular fractions thereof, to inflamed tissue, e.g., living animaltissue in a wound. In some embodiments, the subject has an ocularcondition that results in inflamed tissue, for example, as in dry eyedisease. In some embodiments, the wound is an ocular wound, a surgicalwound, an incision, or an abrasion. The ocular wound can be, forexample, a corneal wound

SDP can thus be used to treat and/or reduce the inflammation caused byconditions such as a wound, infection, or disease. Examples of suchconditions include ocular wounds, surgical wounds, incisions, orabrasions. In some cases, the inflammation is caused by an ocularcondition, such as, dry eye disease or syndrome, corneal ulcer, cornealerosion, corneal abrasion, corneal degeneration, corneal perforation,corneal scarring, an epithelial defect, keratoconjunctivitis, idiopathicuveitis, corneal transplantation, age-related macular degeneration (AMD,wet or dry), diabetic eye conditions, blepharitis, glaucoma, ocularhypertension, post-operative eye pain and inflammation, posteriorsegment neovascularization (PSNV), proliferative vitreoretinopathy(PVR), cytomegalovirus retinitis (CMV), endophthalmitis, choroidalneovascular membranes (CNVM), vascular occlusive diseases, allergic eyedisease, tumors, retinitis pigmen-tosa, eye infections, scleritis,ptosis, miosis, eye pain, mydriasis, neuralgia, cicatrizing ocularsurface diseases, ocular infections, inflammatory ocular diseases,ocular surface diseases, corneal diseases, retinal diseases, ocularmanifestations of systemic diseases, hereditary eye conditions, oculartumors, increased intraocular pressure, herpetic infections, ptyrigium(scleral tumor), wounds sustained to ocular surface,post-photorefractive keratotomy eye pain and inflammation, thermal orchemical burns to the cornea, scleral wounds, keratoconus andconjunctival wounds. In some embodiments, the inflammation and/or ocularcondition is caused by aging, an autoimmune condition, trauma,infection, a degenerative disorder, endothelial dystrophies, and/orsurgery. In one specific example, SDP is used in a formulation to treatdry eye syndrome.

Thus, in various embodiments, SDP and/or fractions thereof such asSDP-4, can be used to inhibit mediators of redox-regulated activation ofthe canonical NF-κB pathway through scavenging of reactive oxygenspecies (ROS), for example hydrogen peroxide, within the cells of theocular environment to reduce the inflammation that causes dry eyesyndrome. Evidence of reduced dry eye symptoms can be a reduction inMMP-9 and TNF-α gene transcription, which are driven by the activationof the NF-κB signaling pathway. Furthermore, MMP-9 enzyme presence inthe cornea tissue will also be reduced.

The following Examples are intended to illustrate the above inventionsand should not be construed as to narrow its scope. One skilled in theart will readily recognize that the Examples suggest many other ways inwhich the inventions could be practiced. It should be understood thatnumerous variations and modifications may be made while remaining withinthe scope of the inventions.

EXAMPLES Example 1. SDP Preparation and The Lawrence Stability Test

Materials. Silkworm cocoons were obtained from Tajima Shoji Co., Ltd.,Japan. Lithium bromide (LiBr) was obtained from FMC Lithium, Inc., NC.An autoclave was obtained from Tuttnauer Ltd., NY. The 3.5 kDamolecular-weight cutoff (MWCO) dialysis membranes were obtained fromThermoScientific, Inc., MA. An Oakton Bromide (Br⁻) double-junctionion-selective electrode was obtained from ISE, Oakton Instruments, IL.

Processing. Two samples, SDP and PASF, were prepared. Briefly, SDP wasproduced by submerging pupae-free, cut silkworm cocoons (3-5cuts/cocoon) into 95° C. heated, deionized water (diH₂O) containing 0.3wt % NaCO₃ at 233 mL water/gram of cocoons. Cocoons were agitated inthis solution for 75 minutes to dissolve sericin, thereby releasing itfrom the silk fibers. The fibers were subsequently washed four times inlike dilutions of diH₂O for 20 minutes per rinse to remove residualsericin. The fibers were then dried in a convection oven at 60° C. for 2hours, weighed, and dissolved in 54 wt % LiBr in water at a ratio of4×LiBr volume per gram of extracted fiber. This solution was covered andthen warmed in a convection oven at 60° C. for 2 hours to expediteextracted fiber dissolution. The solution was then placed in anautoclave and exposed to sterilization conditions (121° C., 17 PSI,97-100% humidity) for 30 minutes to facilitate fibroin transformation.The resulting fibroin solution was allowed to cool to room temperature,then diluted to 5% with diH₂O and dialyzed to remove LiBr salts using a3,500 Da MWCO membrane. Multiple exchanges were performed in diH₂O untilBr⁻ ions were less than 1-ppm as determined in the hydrolyzed fibroinsolution read on an Oakton Bromide (Br⁻) double-junction ion-selectiveelectrode. The solution was then further filtered using a 1-5 μmporosity filter followed by filtration through a 0.2 μm polishingfilter.

A ‘control’ silk fibroin solution was prepared to provide the ‘PASFSolution’. Except the autoclave step, the same process was performed asdescribed above. A sampling volume (5 mL) from each sample was placed inseparate 20 mL glass beakers and the beakers were sealed with foil. Thesamples were then subjected to the Lawrence Stability Test.

The Lawrence Stability Test is performed by placing the aqueous proteintest solution (5% w/v, 50 mg/mL) within the autoclave chamber. Theautoclave is then activated for a cycle at 121° C., 17 PSI, for 30minutes, at 97-100% humidity. After completion of the cycle, thesolution is allowed to cool and is then removed from the autoclavechamber. The solution is then shaken to observe solution gelationbehavior. If the solution has gelled upon shaking for ˜10 seconds, thesample fails the Lawrence Stability Test. Failing the test indicatesthat the material is inherently unstable as a protein solution.

The Lawrence Stability Test was performed on both the SDP solution andthe PASF solution. The PASF solution sample gelled immediately andtherefore failed the Lawrence Stability Test. Conversely, the SDPsolution sample remained in solution indefinitely and therefore passedthe Lawrence Stability Test. The lack of gelation can be attributed tothe fact that the SDP solution production incorporates theautoclave-processing step under the conditions described above.

Example 2. SDP Molecular Weight Characterization

To evaluate the effect of processing on the molecular weightdistribution of solubilized protein, SDP Solution and PASF Solution weresubjected to polyacrylamide gel electrophoresis (PAGE), which separatesproteins by molecular weight. Specifically, 15 μg of each sample wasmixed with running buffer containing sodium dodecyl sulfate anddithiothreitol (Biorad Inc., CA) to remove any secondary foldingstructures and disulfide bonds, respectively. The mixtures were thenheated to 70° C. for 5 minutes. The mixtures were loaded along with a2.5-200 kDa molecular weight ladder (Life Technologies, CA) ontopre-cast, 4-12% polyacrylamide gradient gels containing Bis-Tris buffersalts (Life Technologies, CA), and then exposed to 120V electric fieldfor 90 minutes on a BioRad PowerPac Power supply (BioRad Inc., CA). Thegels were then removed and placed in Coomassie Blue stain for 12 hoursto stain proteins, followed by 6 hours of washing in diH₂O. The gelswere then scanned on a Biorad GS-800 Calibrated Desitometer (BioRadInc., CA).

The results show that the processing employed to prepare the SDPsolution significantly shifts the weight average molecular weight from97 kDa for native fibroin (PASF) to about 53 kDa for SDP. The shift inmolecular weight clearly indicates a transformation of the primary andsecondary structure of the original native fibroin and break-up of thepeptide chains via terminal amide-forming cleavages. In addition, thefibroin light chain of fibroin is not present in the SDP after theautoclaving process (visible at 23-26 kDa in Lane 2 for the prior artfibroin), which indicates that the fibroin light chain portion of theprotein has been degraded or removed by the processing. These resultsdemonstrate that the autoclave processing transforms the native fibroinprotein to a new material that has smaller peptide fragments than thenative fibroin protein. The process further degrades/modifies thefibroin light chain. These transformations result in an SDP materialthat possesses enhanced solution stability as a result of these chemicalchanges.

Further analysis of SDP shows that the average molecular weight of thecomposition is about 53 kDa. Furthermore, about 77% of the peptidechains of SDP are within the range of 10-100 kDa, about 73% of thepeptide chains of SDP are within the range of 10-85 kDa, about 66% ofthe peptide chains of SDP are within the range of 15-85 kDa, about 49%of the peptide chains of SDP are within the range of 20-60 kDa, andabout 31% of the peptide chains of SDP are within the range of 25-50kDa.

Example 3. SDP Stability Study

To further determine the functional impact of the autoclave process onthe stability of the resulting SDP compared to the stability of priorart fibroin, the samples were analyzed using the methods of Wang et al.(Biomaterials 2008, 29(8):1054-1064) to mimic a well-characterized modelof silk fibroin protein gelation. Volumes of both samples (0.5 mL, SDPand PASF) were added to 1.7 mL clear centrifuge tubes and subjected tosonication (20 kHz, 15 seconds). The clear tubes containing thesolutions were then visually monitored for gel formation as a screen forgelation.

The SDP Solution samples failed to form gels, demonstrating enhancedstability. Even 3-months post-sonication, the SDP samples remained insolution and lacked protein aggregation as determined by visualinspection. The PASF Solution sample gelled rapidly (within 2 hours)following sonication. These results further indicate that the autoclaveprocess transforms native isolated fibroin into a new material andinduces stability to the resulting SDP material.

Example 4. In Vitro Analysis of NF-κB Cell Signaling Pathway in HumanCorneal Limbal Epithelium (hCLE) Cultures

The p65 assay as described by Lan et al. (Nuclear Factor-κB: CentralRegulator in Ocular Surface Inflammation and Diseases, The OcularSurface, 10, 137-148 (2012)) was utilized to assess the potentialanti-inflammatory activity of SDP on hCLE cell cultures. As describedearlier, the nuclear transcription factor p65 is part of the NF-κBcomplex, which translocates into the cell nucleus upon activation tofacilitate proinflammatory gene expression, including TNF-α and MMP-9.To assess p65 activity in vitro, confluent hCLE cultures were treatedwith either PBS or PBS containing the potent inflammatory cytokineTNF-α, an autocrine mediator of the NF-κB pathway. Cells were thentreated with PBS or PBS containing 0.1% or 1.0% SDP, respectively.Staining of p65 was localized primarily to the cytoplasm for untreatedcontrols, which is expected for cells in a non-inflammatory state (FIG.1A). However, p65 staining was confined to the nucleus for cellschallenged with TNF-α in the culture medium, indicating that activationof NF-κB inflammatory pathway had taken place (FIG. 1B). Interestingly,p65 staining for SDP-treated cells was largely confined to the cytoplasmand demonstrated a dose-dependent sequestration whereby less nuclearlocalization was exhibited with cells dosed with higher SDPconcentrations (FIG. 1C-D). These results indicate that the SDP proteininhibits the NF-κB inflammatory response in human corneal epithelium invitro.

The inhibition of the NF-κB inflammatory signaling pathway by SDP wasfurther investigated through characterizing TNF-α and MMP-9 geneexpression, which are known to be upregulated during NF-κB-driveninflammation processes. More specifically, increased gene expression ofTNF-α and MMP-9 is mediated by activation of NF-κB, and are biomarkersfor inflammatory cell signaling pathways. Gene expression was measuredusing qPCR on hCLE cultures that were pre-incubated with both PBS andTNF-α cytokine, and then subsequently treated with and without 0.5%wt./vol. SDP as above described. It was observed that the addition ofSDP caused no change in basal gene expression of TNF-α and MMP-9 (FIG.2). However, stimulation with TNF-α evoked a significant rise inexpression of both genes (FIG. 2), which replicates the human NF-κBdriven inflammation cascade. Importantly, treatment with SDP at the timeof TNF-α stimulation evoked a ˜6-fold reduction in expression of bothTNF-α and MMP-9, thereby demonstrating a potent anti-inflammatory effectof SDP on TNF-α-mediated NF-κB gene expression. These resultscorroborate with the previous p65 assay results, collectively supportingthat SDP inhibits NF-κB activation, and as a result inhibitsproinflammatory gene expressing (viz., TNF-α and MMP-9).

Next, studies were carried out in a rabbit corneal epithelial injurymodel to evaluate whether the anti-inflammatory effects of SDP could beextended in vivo. Rabbits were subjected to surgical denudement of thecorneal epithelium to instigate acute inflammatory cascades, and thentreated with eye drops of PBS, PBS plus 0.5%, or PBS plus 2% SDP over 72-hours with 6-hour dosing frequency at approximately 50 μL dropletvolume until complete epithelial healing had occurred. Explanted tissuewas then cryosectioned and immunostained with antibodies against MMP-9protein. The native, non-wounded rabbit cornea exhibited minimal MMP-9expression as anticipated (FIG. 3A), given that reduced presence ofstaining indicates minimal inflammation is taking place as described byKaufmann (The Practical Detection of MMP-9 Diagnoses Ocular SurfaceDisease and May Help Prevent Its Complications, Cornea, 32(2), p 211-216(2013)). However, corneal denudement followed by PBS treatment showedrobust MMP-9 expression throughout the entire epithelial layer, andindicated a high degree of inflammation had occurred (FIG. 3B).

Interestingly, a dose-dependent reduction in rabbit corneal MMP-9expression was observed with the use of eye drops containing SDP (FIG.3C and 3D). Specifically, MMP-9 immunostaining was significantly reducedup to 4-fold for corneas treated with 2% SDP (FIG. 3E). Importantly,attenuated MMP-9 expression did not compromise the integrity of theprotective corneal epithelial layer, evidenced by a robust stratifiedcorneal epithelium with SDP treatment. These data indicate the impactthat SDP treatment has to reduce inflammation within the cornealepithelial tissue post-injury, evidenced by the reduction in MMP-9 withincreasing SDP concentration. Furthermore, these results demonstrate theeffective anti-inflammatory capacity of SDP within a living animaltissue environment, and corroborate previous in vitro studies.

To further bolster the in vivo anti-inflammatory effects of SDP, qPCRwas performed on reverse transcribed, total RNA extracted from therabbit corneal epithelium 72-hours post injury. Specifically, two keybiomarkers of inflammatory signaling, cytokines interleukin (IL)-1β andIL-6, were assessed. Expression of both IL-1β and IL-6 was reducedsignificantly in the presence of SDP treatment (FIG. 4). There was arespective 75% and 95% reduction in gene expression for both SDPconcentrations when compared to PBS-treated control animals. Thesefindings demonstrate the capacity of SDP to inhibit inflammatory geneexpression in vivo, and further substantiate the above in vivo and invitro data. Taken together, experimental evidence shows that SDPinhibits inflammatory processes in vivo, which appears to be directlyrelated to the inhibition of NF-κB inflammatory signaling pathwayactivation by the presence of SDP.

Materials and Methods

SDP production. Bombyx mori silkworm cocoons were purchased from TajimaShoji Co. (Yokohama, Japan). The silk solution was prepared from a batchof 5 g of cocoons that were cut into three pieces each. The cocoons wereboiled in 2 L of 0.03M Na₂CO₃ (Sigma-Aldrich) for 45 minutes to removethe sericin protein. After four rinses in deionized water the extractedsilk fibroin fibers were dried at room temperature overnight. The driedsilk fibroin fibers were then dissolved in a concentrated solution of9.7 M LiBr solution (Sigma-Aldrich) for 2 hours at 60° C. Then, thesolution was autoclaved at and 121° C. under 15 PSI for 30 minutes toexecute the SDP chemical transformation. The autoclaved SDP solution wasthen dialyzed against an approximately 200× volume of water usingSnake-Skin dialysis tubing (Thermo Fisher Scientific, Inc.) with a 3,500molecular weight cut-off (MWCO) for 48-hours and six water exchanges at1, 4, 8, 12, 12, and 12 hour intervals. The dialyzed solution was nextcentrifuged twice at 10,000×g for 20 minutes to remove impurities bydecanting the supernatant each time. Protein concentration was thencalculated by measuring the weight loss on drying of 1 mL samples of SDPsolution (n=3). The solution was finally diluted to a 5 wt./vol. % (50mg/mL) concentration using sterile water and stored at 4° C. until use.

Human corneal epithelial cell culture. Human corneal limbal epithelial(hCLE) cells were thawed from storage in liquid nitrogen and culturedfor 72 hours in keratinocyte-SFM medium (K-SFM, Thermo FisherScientific, Inc.) supplemented with 0.2 ng/mL mouse epithelial growthfactor (EGF, Thermo Fisher Scientific, Inc.), bovine pituitary extract(BPE, Thermo Fisher Scientific, Inc.), 1% penicillin-streptomycin (P/S,VWR, Inc.) and 0.1% CaCl₂.2H₂O (Thermo Fisher Scientific, Inc.).Standard cell culture conditions (37° C., 5% CO₂, >95% humidity) wereused during routine passages.

hCLE p65 staining for NF-κB activation and fluorescence microscopyanalysis.

hCLE cells were grown to ˜80% confluency with a 25,000-cells per wellseeding density. hCLEs were cultured with DMEM/F12 Media in a glassbottom 24-well plate. Human recombinant TNF-α (PeproTech, London, UK)was supplemented at 10 ng/mL and 100 ng/mL for stimulated cultures overa 12-hour challenge. SDP was added to selected cultures at 1 mg/mL and10mg/mL concentration. At the completion of the experiments cultureswere fixed using freshly made 4% paraformaldehyde in phosphate bufferedsaline (PBS). Human p65 antibody (Anti-NF-κB p65, ab16502, Abcam,Cambridge, UK) was added at 1:200 dilution in 1% BSA and 0.1% Tween inPBS and incubated overnight at 4° C. A secondary antibody reactive toanti-rabbit (Alexa Fluor 546, Thermo Fisher Scientific, Inc.) was addedat a 1:500 dilution in PBS. In addition, DAPI nuclear stain (ThermoFisher Scientific, Inc.) was added at a 1:10,000 dilution in PBS.

Fluorescent images were taken using a 63× objective utilizing a 1.6Optivar optic. Z-stack images (10-25 layer range) were captured at 0.25μm slices using a Texas Red filter channel. Image deconvolution wasperformed on each z-stack using 3D Huygens Deconvolution Software(Scientific Volume Imaging BV, The Netherlands) to assist with reducingbackground fluorescence. A total of 40 iterations were performedemploying the software's classic maximum likelihood estimation algorithmfor each z-stack, as it was found that increasing the number ofiterations had a minimal effect on improving image quality. All othersettings were left at the manufacturer's default settings. Images wereproduced using maximum intensity projection (MIP) algorithm included inthe software, where MIP threshold levels were first determined bydefault manufacturer's settings for control corneal tissue to establisha relative fluorescent intensity threshold for each channel.

TNF-α stimulated inflammation assay and gene expression analysis byquantitative polymerase chain reaction (qPCR). hCLE cells were seeded in35 mm dishes and grown to ˜80% confluency before they were dosed witheither PBS or PBS plus 1 ng/mL of recombinant human TNF-α. The cultureswere then incubated at 37° C. for 6 hours. The media was then aspiratedand the cells were washed with warm 1×PBS before they were treated for 6hours with concentration-matched SDP fractions at a 5 mg/mLconcentration, while control groups were dosed with like volumes of PBS.After the defined time had elapsed, total RNA was harvested from thecells using Qiagen RNeasy Plus Mini Kit (Qiagen, Valencia, Calif., USA),and RNA integrity and quantity were verified using electrophoresis andflow cytometry (2100 Bioanalyzer, Agilent Technologies, Santa Clara,Calif.), and UV absorption (Nanodrop Spectrophotometer, ThermoScientific). Afterwards, 450 ng of total RNA from each sample wasreverse transcribed into cDNA using the High Capacity cDNA ReverseTranscription kit (Applied Biosystems, Life Technologies, Grand Island,N.Y.).

Quantitative PCR (qPCR) was carried out in a StepOne Plus real time PCRsystem (Applied Biosystems, Life Technologies, Grand Island, N.Y.) usingthe SYBR Select Master Mix kit (Applied Biosystems, Life Technologies,Grand Island, N.Y.). Genetic expression was performed on total RNAharvested from cells that were not stimulated with TNF-α, as a negativecontrol for the inflammatory stimulus (Native). The expression ofcandidate genes was normalized against the endogenous control geneβ-actin. Relative quantitation was performed using the 2^((−ΔΔCt))method, where 3 experiments were run for each condition each containingthree biological triplicates per condition (N=3, n=3). The populationmean was obtained by averaging the means from each experiment, and apooled standard deviation was calculated for each group. Statisticalcomparison was performed between groups using dCt values by firstperforming a one-way ANOVA followed by post-hoc t-tests to determinep-values using Excel Software (Ver. 14.6.7, Microsoft, Inc.) andStatPlus:mac LE software (Ver. 6.1.5.1, AnalystSoft, Inc., Walnut,Calif.). The following specific primer sets were used for β-Actin, TNF-αand MMP-9 (received from Integrated DNA Technologies, Inc., Coralville,Iowa):

hβ-Actin-Forward: (SEQ ID NO: 2) 5′-AATGTGGCCGAGGACTTTGATTGC-3′hβ-Actin-Reverse: (SEQ ID NO: 3) 5′-AGGATGGCAAGGGACTTCCTGTAA-3′hTNF-α-Forward: (SEQ ID NO: 4) 5′-GAGGCCAAGCCCTGGTATG-3′ hTNF-α-Reverse:(SEQ ID NO: 5) 5′-CGGGCCGATTGATCTCAGC-3′ hMMP-9-Forward: (SEQ ID NO: 6)5′-TGTACCGCTATGGTTACACTCG-3′ hMMP-9-Reverse: (SEQ ID NO: 7)5′GGCAGGGACAGTTGCTTCT-3′

Rabbit corneal injury model, immunohistochemical fluorescent imaginganalysis, and qPCR gene transcription analysis. All animals were handledaccording to the ARVO Statement for the Use of Animals in Ophthalmic andVisual Research, under protocols approved by Institutional Animal Careand Use Committee. Twelve 8-10 week old New Zealand white rabbits wereused to evaluate the capability of SDP to reduce MMP-9 production invivo. Rabbits were anesthetized with intramuscular injections of 35-50mg/kg ketamine, 5-7.5 mg/kg xylazine, and 0.75 mg/kg acepromazine.Topical proparacaine 0.5 wt./vol. % eye drops were also used assupplemental anesthesia. A #15 Bard-Parker blade was then used to remove7 mm of the central corneal epithelium to create a void in theepithelial surface.

Subsequently, the rabbits were divided into three treatment groups,where the wounded corneal surface was treated with either 200 μL ofsterile phosphate buffered saline (PBS, pH 7.4, vehicle treatment), 5mg/mL (i.e., 0.5%) or 20 mg/mL (i.e., 2%) SDP solution in PBS. Thetreatments were administered topically to the wounded eyes, along withtopical moxifloxacin antibiotic drops (Vigamox, Alcon, Inc.),immediately following surgery and subsequently at 6-hour intervals untilcomplete epithelial closure had occurred. Throughout the healingprocess, rabbits were closely monitored for evidence of distress orinfection, and epithelial wound closure was examined every 6 hours byapplying 50 μL of topical fluorescein solution (Sigma-Aldrich) to theinjured cornea and imaging the wound using slit lamp photography undercobalt blue illumination.

Animals from each treatment group were sacrificed immediately afterwound healing was completed (72 hours post-surgery), using an overdoseof pentobarbital (150 mg/kg) administered into the ear vein, and thecorneas from each treatment group were enucleated and excised. For thefirst three rabbits, the healed epithelial surface was removed and thetotal RNA was extracted using the Trizol-chloroform method (ThermoFisherScientific, Inc.). Total RNA from each sample was reverse transcribedinto cDNA using the High Capacity cDNA Reverse Transcription kit(Applied Biosystems, Life Technologies, Grand Island, N.Y.). The cDNAwas then frozen at −80° C. until use.

The remaining three rabbits had extracted corneas fixed immediately in 2wt./vol. % paraformaldehyde for 40 minutes (Electron MicroscopySciences, Hatfield, Pa.). Corneas from the contralateral eyes that didnot undergo surgical denudement were also harvested and fixed, to serveas negative controls for the wound healing process. The fixed corneaswere subsequently washed three times in PBS for 5 minutes each, and thenplaced in 30 wt./vol. % sucrose overnight at 4° C. before embedding inTissue-TEK O.C.T (Sakura Finetek USA Inc., Torrance, Calif., USA) andfrozen at −80° C. for cryo-sectioning. Ten-micron thick cross-sections,through the center of the cornea, were obtained and mounted onSuperfrost-plus glass slides (Thermo Fisher Scientific, Inc.) forimmunohistochemical staining and analysis. Samples were washed threetimes in PBS and then incubated in blocking buffer containing 1 wt./vol.% BSA (Sigma-Aldrich), 0.25 wt./vol. % Triton-X-100 (Sigma-Aldrich), and2.5 wt./vol. % goat serum in 1×PBS, for 1 hour at room temperature.After blocking, samples were incubated with murine primary antibodysolutions (1:100) for MMP-9 (ab58803, Abcam PLC, Cambridge, UK)overnight at 4° C.

Subsequently, the samples were rinsed thoroughly with PBS and thenincubated with Alexa Fluor 488 Green goat anti-mouse secondary antibody(ab150113, Abcam PLC, Cambridge, UK) at a 1:500 dilution for 1 hour atroom temperature, protected from light. Samples were also stained withAlexa Fluor® 568 phalloidin (Thermo Fisher Scientific, Inc.) at a 1:200dilution for 20 minutes at room temperature and protected from light tostain for actin cytoskeletal structure. After washing with PBS, Sampleswere mounted with VECTASHIELD Mounting Medium with DAPI (VectorLaboratories, Burlingame, Calif., USA) to stain for cell nuclei, andcovered with a glass coverslip before imaging.

Fluorescent images were taken using a 63× objective utilizing a 1.6Optivar optic. Z-stack images (10-25 layer range) were captured at 0.25μm slices using the green fluorescent protein (GFP) filter channel.Image deconvolution was performed on each z-stack using 3D HuygensDeconvolution Software (Scientific Volume Imaging BV, The Netherlands)to assist with reducing background fluorescence. A total of 40iterations were performed employing the software's classic maximumlikelihood estimation algorithm for each z-stack, as it was found thatincreasing the number of iterations had a minimal effect on improvingimage quality. All other settings were left at the manufacturer'sdefault settings. Images were produced using maximum intensityprojection (MIP) algorithm included in the software, where MIP thresholdlevels were first determined by default manufacturer's settings forcontrol corneal tissue to establish a relative fluorescent intensitythreshold for each channel. Then, native and SDP-treated cornea groupswere imaged using these same threshold settings to allow for groupcomparisons (N=3, n=3) of fluorescent image intensities.

Next, fluorescence intensity of each image was measured using ImageJsoftware (NIH, Ver. 1.48, NIH) by subtracting the mean integrated colordensities of a non-fluorescing region from the traced fluorescent regionto eliminate background. Fluorescent intensity values among thedifferent groups were then calculated. Groups were then statisticallycompared through one-way ANOVA analysis followed by ad hoc t-tests todetermine p-values using Excel Software (Microsoft, Inc., Ver. 14.6.7)and StatPlus:mac LE software (AnalystSoft, Inc., Ver. 6.1.5.1).

Quantitative PCR (qPCR) was carried out on an ABI 7000 real time PCRsystem (Applied Biosystems, Life Technologies) using the SYBR SelectMaster Mix kit (Applied Biosystems, Life Technologies). Geneticexpression was performed on produced cDNA from the harvested from therabbit corneal epithelium. The expression of candidate genes wasnormalized against the endogenous control gene β-actin. Relativequantitation was performed using the 2^((−ΔΔCt)) method. Statisticalcomparison was performed between groups using dCt values by firstperforming a one-way ANOVA followed by post-hoc t-tests to determinep-values using Excel Software (Microsoft, Inc., Ver. 14.6.7) andStatPlus:mac LE software (AnalystSoft, Inc., Ver. 6.1.5.1). Thefollowing specific primer sets were used for β-Actin, IL-1β and IL-6genes (received from Integrated DNA Technologies, Inc., Coralville,Iowa):

rβ-actin-Forward: (SEQ ID NO: 8) 5′-GCTATTTGGCGCTGGACTT-3′rβ-actin-Reverse: (SEQ ID NO: 9) 5′-GCGGCTCGTAGCTCTTCTC-3′rIL-1β-Forward: (SEQ ID NO: 10) 5′-TTGAAGAAGAACCCGTCCTCTG-3′rIL-1β-Reverse: (SEQ ID NO: 11) 5′-CTCATACGTGCCAGACAACACC-3′rIL-6-Forward: (SEQ ID NO: 12) 5′-CTACCGCTTTCCCCACTTCAG-3′rIL-6-Reverse: (SEQ ID NO: 13) 5′-TCCTCAGCTCCTTGATGGTCT-3′

Example 5. SDP and SDP-4 Inhibit Hydrogen Peroxide-Mediated RedoxSignaling

Electron Paramagnetic Resonance (EPR) spectroscopy was used toselectively quantify concentrations of hydrogen peroxide (H₂O₂) insolution. Specifically, 20 μM of H₂O₂ was added to aqueous solutionscontaining 0, 0.5, 1.0, or 5.0% of PASF, SDP, or SDP-4, and wasincubated at room temperature for 24 hours. To quantitate remaining H₂O₂levels following incubation, 200 μM of the H₂O₂-specific spin probe1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine (CMH) wasthen added along with assay reagents 4-acetamidophenol (AAP, 1 mM),diethylenetriaminepentaacetic acid (DTPA, 200 μM), and horseradishperoxidase (HRP, 1 U/mL). This mixture was then incubated at 37° C. for30 minutes, during which time AAP is oxidized by H₂O₂ in the presence ofHRP to generate phenoxyl radicals, which then react with the CMH spinprobe to generate a CM radical, which is detected and quantified by EPR.

Results: PASF elevated EPR signal amplitude above control levels withincreasing protein concentration, indicating that PASF oxidizes the H₂O₂spin probe directly. In contrast, addition of SDP evoked aconcentration-dependent reduction in EPR signal amplitude, demonstratingthat SDP proteins scavenge H₂O₂ by 40% at 5.0% SDP. These reductionswere even more robust in the presence of SDP-4 proteins, whereby 5.0%SDP-4 reduced H₂O₂ levels by over 80%. See FIG. 5.

The capacity of SDP and more so SDP-4 to scavenge H₂O₂ and inhibit redoxsignaling is tyrosine-driven. Tyrosine is a known long-term actingantioxidant due to its aromatic and hydroxyl-containing functional groupwhich permit ease of electron shuffling inherent to redox signaling (seeVan Overveld et al., Chemico-Biological Interactions, 127(2000),151-161). SDP and SDP-4 possess high tyrosine composition (greater thanor equal to about 13% wt./wt.), and these proteins enhance tyrosinedelivery to the ocular surface. The physiologic solubility of tyrosineis 0.4 mg/mL, yet tyrosine solubility in 1% wt./vol. SDP over threetimes greater (1.3 mg/mL), thus providing a proportional increase per 1%wt./vol. of SDP. Furthermore, the aqueous solubility of SDP and SDP-4exceeds 80% wt./vol., far greater than other known proteins.

Example 6. Fractionation and Molecular Weight Distribution of SDPProtein Solutions

Fractionation of a regenerated SDP solution was accomplished through aseries of centrifugation steps utilizing Amicon Ultra 15 mL centrifugalfilters of 100, 50, 30, and 10 kDa MW cutoffs (EMD-Millipore, MA, USA).To evaluate the molecular weight range of the fractionated SDPsolutions, the electrophoretic mobility of the SDP protein wasvisualized using SDS-PAGE and compared to that of unfractionated SDPsolution. SDS-PAGE of the unfractionated SDP indicated a wide molecularweight distribution of SDP protein within the solution, as evidenced bya large smear located approximately between the 300 kDa and 30 kDamolecular mass ranges.

Fractionation of the regenerated SDP solution produced four fractionsranging from high to low molecular weight SDP proteins (SDP-1, SDP-2,SDP-3, and SDP-4, respectively). See FIG. 6. When compared tounfractionated SDP solution, SDS-PAGE of the high molecular weightsolution produced a smear indicating an approximate molecular weightdistribution between the 300 kDa and 100 kDa range (SDP-1 and SDP-2),while the low molecular weight solution produced a smear indicating amolecular weight distribution predominantly in the 30 kDa range (SDP-3and SDP-4), and thus confirming the fractionation of SDP into high andlow molecular weight SDP protein solutions.

For example, a 50 mg/mL aqueous SDP solution derived from Bombyx morisilkworm cocoons was used for the following study. Fractionation of SDPprotein fragments was accomplished using Amicon Ultra 15 mL centrifugalfilters of 100, 50, 30, and 10 kDa MW cutoffs (EMD-Millipore, MA, USA).Briefly, 15 mL of a 40 mg/mL SDP stock solution was added to acentrifugal filter with 100 kDa MW cutoff and spun down at 4,000×g for30 minutes for isolation of SDP protein fragments of 100 kDa MW andabove. The isolated concentrate was collected and the filtrate wassubsequently transferred to a centrifugal filter with 50 kDa MW cutoffand spun down again at 4,000×g for 30 minutes to isolate SDP proteinfragments of ˜50 kDa MW. The isolated concentrate was collected and thefiltrate was then transferred to a centrifugal filter with 30 kDa MWcutoff and spun down again at 4,000×g for 30 minutes to isolate SDPprotein fragments of ˜30 kDa MW. The isolated concentrate was collectedand the filtrate was then transferred to a centrifugal filter with 10kDa MW cutoff and spun down again at 4,000×g for 30 minutes to isolateSDP protein fragments of ˜10 kDa MW. The collected concentrates fromeach MW cutoff were individually washed, 6 times, with 5 mL of dH₂O andspun down again at 4,000×g for 30 minutes using centrifugal filters withthe respective MW cutoff filter size for each concentrate. Fractionationof SDP protein fragments was verified using SDS-PAGE (FIG. 6) andCoomassie blue R-250 staining (Gibco, Invitrogen Corporation, GrandIsland, N.Y.).

ImageJ analysis of the SDP-4 fraction showed that SDP-4 has an averagemolecular weight of 34 kDa, as determined by SDS-PAGE. Similarfiltration of PASF (30 kDa MWCO filter) provided a lower molecularweight fraction having an average molecular weight of 51 kDa, and aseparate higher average molecular weight fraction (90 kDa). Furtheranalysis of SDP fractions and PASF fractions is summarized in the tablebelow.

SDP-4 SDP-1-3 SDP PASF-4 PASF-1-3 PASF kDa 100-10  85.3% 76.2% 77.3%81.1% 52.8% 50.2% Range 85-10 83.6% 71.5% 73.0% 77.4% 48.4% 45.2% 85-1572.1% 66.7% 66.2% 71.3% 44.8% 41.6% 60-20 57.7% 49.1% 48.5% 53.4% 31.6%27.1% 50-25 39.0% 31.4% 30.8% 34.6% 20.1% 15.7% Average 34 57 53 51 9097 MW (kDa)

Fractions having average molecular weights of less than 10 kDa areunstable in solution and form gels within 1-2 hours and are thereforetypically removed from the SDP compositions and fractions.

Example 7. SDP Stability Study of SDP-4 and SDP-1-3

The stability study described in Example 3 was also performed on SDP-4and a low average molecular weight fraction of isolated native fibroin(PASF-4). Sonication-induced secondary structure formation and gelationwas found to be absent in the SDP-4 solution after sonication challenge.The SDP-4 solution remained clear and free flowing. The lack of gelationindicates the significantly greater protein stability of SDP-4, whereasthe PASF-4 solution gelled within 2 hours after sonication challenge,indicating its instability in solution. SDP-4 remained in solutionthroughout the time course of the experiment (96 hours).

The stability study was also carried out on higher molecular weightfractions of SDP (equivalent to the combination of SDP-1, SDP-2, andSDP-3; referred to as SDP-1-3). The aqueous solution of SDP-1-3 remaineda free-flowing solution throughout the time course of the experiment(more than 24 hours). However, the higher molecular weight fractions ofisolated native fibroin (PASF-1-3) gelled within 15 minutes, indicatingsecondary protein structure formation and hence instability.

Experimental conditions: 1 mL of 4% wt./wt. solutions of SDP-4, SDP-1-3,PASF-4, and PASF-1-3 were subjected to sonication at 60% amplitude, 20Hz pulse frequency, for 3 minutes. Solutions were then monitored at roomtemperature until gelation had occurred for PASF-4 and PASF-1-3. SDP-4and SDP-1-3 remained in solution for more than 96 hours and 24 hours,respectively (the time course of this study).

Example 8. Enhanced Wound Healing Properties of SDP-1 and SDP-2

Wound healing was evaluated on confluent hCLE monolayers subjected to ascratch assay in the absence or presence of SDP fractions (10 mg/mL).Wound closure rates were evaluated using time-lapse microscopy.Proliferation of hCLEs treated with SDP or PBS controls were evaluatedby MTT assay.

SDP MW had a critical impact on the behavior of injured hCLE cultures.Low average M.W. fractions of <100 kDa (i.e., SDP-3 and SDP-4)significantly accelerated repopulation of denuded (scratched) hCLE cellsvs. PBS treated control cultures by 6 hours, which persisted untilconfluency (16 hours vs. 20 hours for controls) (FIGS. 7 and 8). SDP-3and SDP-4 significantly increased hCLE proliferation vs. controlcultures treated with PBS, as evidenced by increased (>50%) metabolicactivity by the MTT assay results. Conversely, high MW fractions of >100kDa (i.e., SDP-1 and SDP-2) inhibited repopulation, althoughpro-proliferative effects of SDP were still observed (FIG. 9). Theseresults demonstrate the enhanced potency effect that SDP-3 and SDP-4fractions have on hCLE cell migration outcomes in vitro.

Example 9. Anti-Inflammatory Properties

Inflammatory properties of SDP fractions 1-4 were evaluated on hCLEcultures stimulated with the pro-inflammatory cytokine tumor necrosisfactor-alpha (TNF-α, 1 ng/mL) in the presence or absence of SDPfractions. qPCR was used to quantitate subsequent expression ofinflammatory genes. Secretion of these proteins by hCLE cells wasevaluated by ELISA. Functional significance of altered inflammatory geneexpression was assessed using a Transwell co-culture assay with TNF-αstimulated hCLE cultures and a promyelocytic immune cell line (HL-60),performed in the presence or absence of SDP fractions.

TNF-α challenged hCLE cultures robustly increased expression ofinflammatory genes TNF-α, interleukins 6 and 1α/β, and protease MMP-9,which expression was significantly attenuated with low MW SDP (FIG. 10).This translated into significant reductions in the secretion of TNF-αand MMP-9 by stimulated hCLEs as measured by ELISA at 8 hours (FIG. 11).TNF-α-challenged hCLEs evoked significant recruitment of HL-60 cellsthat was normalized by the addition of SDP-4, demonstrating a functionalrelationship between impaired inflammatory signaling and downstreamimmune responses in vitro (FIG. 12).

Example 10. Preparation of OTC and Anti-Inflammatory Eye DropFormulations

An eye drop composition can be prepared to take advantage of thetherapeutic properties of SDP to treat the ocular system because ofdisease or injury. SDP molecules can be optionally isolated based onmolecular weights (e.g., SDP-1, SDP-2, SDP-3, SDP-4, or a combinationthereof), or used as a whole composition (e.g., SDP). A composition ofprotein molecules of low average molecular weight, such as less thanabout 40 kDa, can be prepared and is referred to as SDP-4. A secondcomposition of protein molecules that includes all molecular weights ofthe SDP composition or molecules more than about 40 kDa can also beprepared. Each composition can include water, at least one buffer orbuffer system (e.g., phosphate buffered saline (PBS), citrate, borate,Tris, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)),optionally at least one preservative (e.g., perborate, benzalkoniumchloride (BAK)) and optionally at least one additional excipient,surfactants, stabilizers or salt (e.g., sulfanilic acid, trehalose,glycerin, ethylenediaminetetraacetic acid (EDTA), polyethylene glycol(PEG), mannitol, polysorbate, sodium chloride (NaCl), magnesium chloride(MgCl₂), calcium chloride (CaCl₂), or lithium bromide (LiBr)).

The eye formulation containing the first compositions above can beapplied as a therapeutic product to a dry eye disease patient, a woundedpatient, or a surgical wound of an otherwise healthy patient (e.g., forpost-refractive or cataract surgery). The disease or injury can bemonitored over time for inflammation and wound closure rate, and forpatient comfort and pain assessment. The second compositions can be usedin over-the-counter products, such as an artificial tears eye dropproduct, as a protein excipient to help with enhancing formulationwetting, spreading, and patient comfort.

An example of an eye drop formulation would contain as low as 0.1%wt./vol. SDP-4 or SDP to as high as 10% wt./vol. SDP-4 or SDP. The SDP-4or SDP material would be dissolved into purified water, where a buffersystem such as citric acid buffer, Tris buffer, PBS buffer, or boratebuffer would be created in a 1 mmol to 1,000 mmol concentration.Additional excipient ingredients may be added to the formulation. Asurfactant, such as polysorbate, could be added in the range of a0.01%-0.1% wt./vol. concentration. Stabilizing sugar molecules can beadded, such as trehalose, dextrose, or sucrose, at concentrationsranging from 10 mmol-500 mmol. Demulcent molecules can be added asocular lubricants, such as PEG, carboxy methyl cellulose, hypromellose,hydroxypropyl methylcellulose, or glycerin, at concentrations rangingfrom 0.1%-2.0% wt./vol. Salts may also be added to reduce molecularinteractions and stabilize the formulation, such as NaCl, MgCl₂, CaCl₂,or LiBr, at concentration ranging from 10 mmol-500 mmol. Amino acidmolecules can be added as stabilizing agents, such as L-glutamine orL-arginine, at concentrations ranging from 10 mmol-500 mmol. Chelatingagents can be added as stabilizing agents, such as EDTA, atconcentrations ranging from 0.01%-0.1% wt./vol. Anti-microbial agentscan be added to the formulation, such as perborate or BAK, atconcentrations of up to 0.015% wt./vol.

Below is a table of a few example base formulations that have beenproduced containing the SDP-4 and/or SDP molecules, in which additionaladditives or excipients can be added to enhance formulation applicationsdescribed above:

Composition Ingredient 1 2 3 4 5 SDP-4 or SDP 5 or 10 g 5 or 10 g 5 or10 g 5 or 10 g 5 or 10 g Phosphate 10 mmol — — — — NaCl 137 mmol — — — —KCl 2.7 mmol — — — — Citric Acid — 82 mmol 8 mmol — — Trisodium Citrate— 18 mmol 92 mmol — — Tris Hydrochloric Acid — — — 7.02 g 0.76 g TrisBase — — — 0.67 g 5.47 g Water 1 L 1 L 1 L 1 L 1 L pH 7.4 3.0 6.2 7.29.0

SDP, or an SDP fraction such as SDP-4, can also be added to known eyeformulations such as commercial and prescription eye drops and ointmentsto improve wetting and patient comfort. Examples of ophthalmic solutionsthat SDP or SDP-4 can be added to include brimonidine tartrate,brimonidine tartrate/timolol maleate, alcaftadine, bimatoprost,cyclosporine, gatifloxacin, ketorolac tromethamine, or lifitegrastophthalmic solutions. Examples of other formulations that SDP or SDP-4can be added to are described in U.S. Pat. Nos. 5,468,743; 5,880,283;6,333,045; 6,562,873; 6,627,210; 6,641,834; 6,673,337; 7,030,149;7,320,976; 7,323,463; 7,351,404; 7,388,029; 7,642,258; 7,842,714;7,851,504; 8,008,338; 8,038,988; 8,101,161; 8,133,890; 8,207,215;8,263,054; 8,278,353; 8,299,118; 8,309,605; 8,338,479; 8,354,409;8,377,982; 8,512,717; 8,524,777; 8,541,463; 8,541,466; 8,569,367;8,569,370; 8,569,730; 8,586,630; 8,629,111; 8,632,760; 8,633,162;8,642,556; 8,648,048; 8,648,107; 8,664,215; 8,685,930; 8,748,425;8,772,338; 8,858,961; 8,906,962; and 9,248,191, and 7,314,938;7,745,460; 7,790,743; 7,928,122; 8,084,047; 8,168,655; 8,367,701;8,592,450; 8,927,574; 9,045,457; 9,085,553; 9,216,174; 9,353,088; and9,447,077.

While specific embodiments have been described above with reference tothe disclosed embodiments and examples, such embodiments are onlyillustrative and do not limit the scope of the invention. Changes andmodifications can be made in accordance with ordinary skill in the artwithout departing from the invention in its broader aspects as definedin the following claims.

All publications, patents, and patent documents are incorporated byreference herein, as though individually incorporated by reference. Nolimitations inconsistent with this disclosure are to be understoodtherefrom. The invention has been described with reference to variousspecific and preferred embodiments and techniques. However, it should beunderstood that many variations and modifications may be made whileremaining within the spirit and scope of the invention.

In accordance with 35 U.S.C. 102(b)(2)(c), (1) the subject matterdisclosed was developed and the claimed invention was made by, or onbehalf of, the parties to a joint research agreement that was in effecton or before the effective filing date of the claimed invention; (2) theclaimed invention was made as a result of activities undertaken withinthe scope of the joint research agreement; and (3) the names of theparties to the joint research agreement are (a) Silk Technologies, Ltd.and (b) Cornell University. The inventors have assigned their rights inthe invention to Silk Technologies, Ltd. or Cornell University.

1. A fibroin-derived protein composition that possesses enhancedstability in an aqueous solution compared to native silk fibroin,wherein: the primary amino acid sequences of the fibroin-derived proteincomposition differ from native fibroin by at least 4% with respect tothe absolute values of the combined differences in amino acid content ofserine, glycine, and alanine; cysteine disulfide bonds between thefibroin heavy and fibroin light protein chains of fibroin are reduced oreliminated; the composition has a serine content that is reduced bygreater than 25% compared to native fibroin protein, wherein the serinecontent is at least about 5%; wherein the average molecular weight ofthe fibroin-derived protein composition is less than about 60 kDa andgreater than about 10 kDa, wherein greater than 50% of the proteinchains of the protein composition have a molecular weight of less about60 kDa, and the composition lacks beta-sheet protein structure in theaqueous solution.
 2. The protein composition of claim 1 wherein greaterthan 50% of the protein chains of the protein composition have amolecular weight within the range of about 25 kDa to about 60 kDa
 3. Theprotein composition of claim 1 wherein about 39% of the protein chainsof the protein composition have a molecular weight within the range ofabout 20 kDa to about 50 kDa.
 4. The protein composition of claim 1wherein the protein composition does not gel upon sonication of anaqueous solution of the protein composition at concentrations of up to10% w/w.
 5. The protein composition of claim 1 wherein the proteincomposition comprises less than 8% serine amino acid residues.
 6. Theprotein composition of claim 1 wherein the protein composition comprisesgreater than 46.5% glycine amino acids.
 7. The protein composition ofclaim 1 wherein the protein composition comprises greater than 30.5%alanine amino acids.
 8. The protein composition of claim 1 wherein theprotein composition completely re-dissolves in water after being driedto a thin film.
 9. The protein composition of claim 1 wherein theprotein composition maintains an optical absorbance in aqueous solutionof less than 0.25 at 550 nm after at least five seconds of sonication.10. An ophthalmic formulation comprising the protein composition ofclaim 1 and water, and optionally one or more of a buffering medium, asalt, a stabilizer, a preservative, and a lubricant.
 11. A method forreducing inflammation comprising administering the fibroin-derivedprotein composition according to claim 1 to inflamed tissue, therebyreducing transcription factor signaling within cell nuclei of thetissue, thereby reducing the inflammation.
 12. The method of claim 11wherein the administration to inflamed tissue reduces transcription ofone or more of the inflammatory genes TNF-α, MMP-9, IL-1β, and IL-6. 13.The method of claim 11 wherein the administration is to the cornea andthe administration reduces the presence of MMP-9 in the cornea.
 14. Themethod of claim 11 wherein the administration is to the eye and theadministration reduces inflammation on the ocular surface.
 15. Themethod of claim 11 wherein the reduction in inflammation is accompaniedby increased cell migration rates at the point of inflammation.
 16. Themethod of claim 11 wherein the protein composition has an averagemolecular weight of less than 40 kDa.
 17. The method of claim 16 whereinthe protein composition has an average molecular weight of about 25 kDato about 38 kDa.
 18. The method of claim 11 wherein the fibroin-derivedprotein composition is dissolved in an ophthalmic formulation comprisingone or more of a buffering medium, a salt, a stabilizer, a preservative,and a lubricant.
 19. The method of claim 11 wherein the inflammation iscaused by an ocular condition, wherein the ocular condition is dry eyesyndrome, corneal ulcer, corneal erosion, corneal abrasion, cornealdegeneration, corneal perforation, corneal scarring, epithelial defect,keratoconjunctivitis, idiopathic uveitis, corneal transplantation,age-related macular degeneration, diabetic eye, blepharitis, glaucoma,ocular hypertension, post-operative eye pain and inflammation, posteriorsegment neovascularization, proliferative vitreoretinopathy,cytomegalovirus retinitis, endophthalmitis, choroidal neovascularmembrane, vascular occlusive disease, allergic eye disease, tumor,retinitis pigmentosa, eye infection, scleritis, ptosis, miosis, eyepain, mydriasis, neuralgia, cicatrizing ocular surface disease, ocularinfection, inflammatory ocular disease, ocular surface disease, cornealdisease, retinal disease, ocular manifestations of systemic diseases,hereditary eye condition, ocular tumor, increased intraocular pressure,herpetic infection, ptyrigium or scleral tumor, wound sustained toocular surface, post-photorefractive keratotomy eye pain andinflammation, thermal or chemical burn to the cornea, scleral wound, orkeratoconus and conjunctival wound.
 20. The method of claim 19 whereinthe inflammation is caused by dry eye syndrome.